TW202216767A - Antibodies binding to cd3 and folr1 - Google Patents

Abstract

The present invention generally relates to bispecific antibodies that bind to CD3 and Folate Receptor 1 (FolR1), e.g. for activating T cells. In addition, the present invention relates to polynucleotides encoding such antibodies, and vectors and host cells comprising such polynucleotides. The invention further relates to methods for producing the antibodies, and to methods of using them in the treatment of disease.

Description

Antibodies that bind to CD3 and FolR1

The present invention generally relates to bispecific antibodies that bind to CD3 and folate receptor 1 (FolR1), eg, for activation of T cells. In addition, the present invention relates to polynucleotides encoding such antibodies, as well as vectors and host cells comprising these polynucleotides. The present invention further relates to methods of producing such antibodies, and methods of using such antibodies to treat diseases.

In various clinical settings, it is often necessary to selectively destroy individual cells or specific cell types. For example, the primary goal of cancer therapy is to specifically destroy tumor cells, while leaving healthy cells and tissues intact.

One attractive way to achieve this goal is by inducing an immune response against the tumor, allowing immune effector cells such as natural killer (NK) cells or cytotoxic T lymphocytes (CTL) to attack and destroy tumor cells. CTLs constitute the most potent effector cells of the immune system, but they cannot be activated by effector mechanisms mediated by the Fc domain of conventional therapeutic antibodies.

In this regard, in recent years, activated invariant components designed to bind with one "arm" to surface antigens on target cells and the second "arm" to the T cell receptor (TCR) complex of bispecific antibodies. Simultaneous binding of such antibodies to their two targets will force a temporal interaction between target cells and T cells, leading to activation of any cytotoxic T cells and subsequent lysis of target cells. Thus, the immune response is redirected to target cells independently of peptide antigen presentation by target cells or specificity of T cells, and is associated with activation of normal MHC-restricted CTLs. In this context, it is important that the CTLs are only activated when the target cells present them with the bispecific antibody, ie, mimic the immune synapse. In particular, the desired bispecific antibodies do not require lymphocyte pretreatment or costimulation to elicit efficient lysis of target cells.

CD3 has been extensively explored as a drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressive therapy in autoimmune diseases, such as type I diabetes, or to treat transplant rejection. The CD3 antibody muromonab-CD3 (OKT3) was the first monoclonal antibody approved for clinical use in humans in 1985.

A more recent application of CD3 antibodies is in the form of bispecific antibodies, which bind CD3 on the one hand and tumor cell antigens on the other hand. Simultaneous binding of such antibodies to their two targets will force a temporal interaction between target cells and T cells, leading to activation of any cytotoxic T cells and subsequent lysis of target cells.

FOLR1 is expressed on epithelial tumor cells of various origins, such as ovarian cancer, lung cancer, breast cancer, kidney cancer, colorectal cancer, endometrial cancer. Several therapeutic antibodies, such as farletuzumab, antibody drug conjugates, or adoptive T cell therapy targeting FOLR1 for tumor imaging have been described (Kandalaft et al., J Transl Med. 2012 8 Sep 3;10:157. doi: 10.1186/1479-5876-10-157; van Dam et al., Nat Med. 2011 Sep 18;17(10):1315-9. doi: 10.1038/nm. 2472; Clifton et al, Hum Vaccin. 2011 Feb;7(2):183-90. Epub 2011 Feb 1; Kelemen et al, Int J Cancer. 2006 Jul 15;119(2):243- 50; Vaitilingam et al, J Nucl Med. 2012 Jul; 53(7); Teng et al, 2012 Aug; 9(8):901-8. doi: 10.1517/17425247.2012.694863. Epub 2012 June Sep 5. Attempts have been made to target folate receptor positive tumors using constructs targeting the folate receptor and CD3 (Kranz et al, Proc Natl Acad Sci US A. 1995 Sep 26;92(20 ): 9057–9061; Roy et al, Adv Drug Deliv Rev. 2004 Apr 29;56(8):1219-31; Huiting Cui et al Biol Chem. 2012 Aug 17;287(34) : 28206–28214; Lamers et al, Int. J. Cancer. 60(4):450 (1995); Thompson et al, MAbs. 2009 Jul-Aug; 1(4):348-56. Epub 2009 July 19, 2008; Mezzanzanca et al., Int. J. Cancer, 41, 609–615 (1988). However, the approaches taken so far have many shortcomings. The molecules used so far cannot be easily and reliably produced because of their Chemical cross-linking is required. Likewise, hybrid molecules cannot be produced on a large scale like human proteins and require the use of rat, mouse, or other highly immunogenic proteins that are highly immunogenic when administered to humans, so therapeutic Limited value. In addition, many existing molecules retain Fc gR binding.

More recently, WO2016/079076 describes T cell activating bispecific antigen binding molecules targeting CD3 and FolR1.

An important requirement that antibodies must meet for therapeutic purposes is adequate stability both in vitro (for drug storage) and in vivo (after administration to a patient).

Modifications such as asparagine deamidation are typical of the degradation of recombinant antibodies and may affect in vitro stability and in vivo biological function.

In view of the great therapeutic potential of antibodies, in particular bispecific antibodies for activating T cells, bispecific CD3/FolR1 antibodies with optimized properties are needed.

The present invention provides antibodies, including multispecific (eg, bispecific) antibodies, that bind to CD3 and are resistant to degradation by, for example, deamidation of asparagine, and are therefore particularly stable as required for therapeutic purposes. The provided (multispecific) antibodies further combine good efficacy and producibility with low toxicity and favorable pharmacokinetic properties.

As shown herein, the antibodies provided by the invention that bind to CD3 (including multispecific binding activity relative to the binding activity after 2 weeks at pH 6, -80°C, as determined by surface plasmon resonance (SPR) Antibody) retained more than about 90% of its binding activity to CD3 after 2 weeks at pH 7.4, 37°C.

In particular aspects, the present invention provides dual binding to CD3 and folate receptor 1 (FolR1) relative to binding activity after 2 weeks at pH 6, -80°C, as determined by surface plasmon resonance (SPR) Specific antibodies, these bispecific antibodies retained more than about 90% of their binding activity to CD3 after 2 weeks at pH 7.4, 37°C.

In one aspect, there is provided a bispecific antibody that binds to CD3 and folate receptor 1 (FolR1), wherein the bispecific antibody comprises (i) a first antigen-binding domain capable of specifically binding to CD3, the first antigen-binding domain comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2 , HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, SEQ ID NO : LCDR 2 of 9 and LCDR 3 of SEQ ID NO: 10; and (ii) a second antigen-binding domain capable of specifically binding to FolR1.

In one aspect, a bispecific antibody is provided, wherein the VH of the first antigen binding domain comprises at least about 95%, 96%, 97%, 98%, 99% or the amino acid sequence of SEQ ID NO:7 100% identical amino acid sequence, and/or VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11 .

In one aspect, the bispecific antibody binds CD3 and FolR1, wherein the bispecific antibody comprises (i) a first antigen-binding domain capable of specifically binding to CD3, the first antigen-binding domain comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11; and (ii) a second antigen-binding domain capable of specifically binding to FolR1.

In one aspect, the first antigen binding domain is a Fab molecule.

In one aspect, the bispecific antibody comprises an Fc domain consisting of a first subunit and a second subunit.

In one aspect, the bispecific antibody comprises a third antigen-binding domain capable of specifically binding to FolR1.

In one aspect, the second antigen binding domain and/or the third antigen binding domain when present is a Fab molecule.

In one aspect, the first antigen binding domain is a Fab molecule in which the variable domains VL and VH of the Fab light and Fab heavy chains or the constant domains CL and CH1, in particular the variable domains VL and VH, are substituted for each other.

In one aspect, the second antigen binding domain and, if present, the third antigen binding domain are conventional Fab molecules.

In one aspect, the second antigen-binding domain and, if present, the third antigen-binding domain are Fab molecules in which, in the constant domain CL, the amino acid at position 124 is lysine (K), arginine ( R) or histidine (H) (according to Kabat numbering) independently substituted, and the amino acid at position 123 is independently lysine (K), arginine (R) or histidine (H) (according to Kabat) numbering) and in constant domain CH1 the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index) and the amino acid at position 213 is substituted with Glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index number) independently substituted.

In one aspect, the first antigen binding domain and the second antigen binding domain are fused to each other, optionally via a peptide linker.

In one aspect, the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and (i) the second antigen-binding domain is C-terminal to the Fab heavy chain and N-terminal to the Fab heavy chain of the first antigen-binding domain terminal fusion, or (ii) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain.

In one aspect, the first antigen-binding domain, the second antigen-binding domain, and, if present, the third antigen-binding domain are each a Fab molecule, and the bispecific antibody comprises a first subunit and a second subunit. and wherein (i) the second antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain. The N-terminus of the first subunit of the Fc domain is fused, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain and the second antigen-binding domain is fused at the N-terminus of the Fab heavy chain of the second antigen-binding domain. The C-terminus of the Fab heavy chain is fused to the N-terminus of the first subunit of the Fc domain; and the third antigen binding domain, when present, is fused to the N-terminus of the second subunit of the Fc domain at the C-terminus of the Fab heavy chain.

In one aspect, the Fc domain is an IgG Fc domain, in particular _an IgGi Fc domain.

In one aspect, the Fc domain is a human Fc domain.

In one aspect, the Fc comprises modifications that facilitate association of the first and second subunits of the Fc domain.

In one aspect, the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function.

In one aspect, the second antigen binding domain and, when present, the third antigen binding domain comprises: a VH comprising HCDR 1 of SEQ ID NO: 124, HCDR 2 of SEQ ID NO: 125, and SEQ ID NO: 126 HCDR 3 of SEQ ID NO: 8; and VL comprising LCDR 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9, and LCDR 3 of SEQ ID NO: 10.

In one aspect, there is provided a bispecific antibody as described herein above, wherein the second antigen binding domain and, when present, the third antigen binding domain comprises: a VH comprising an amine group with SEQ ID NO: 123 An amino acid sequence whose acid sequence is at least about 95%, 96%, 97%, 98%, 99% or 100% identical; and/or a VL comprising at least about 95% the amino acid sequence of SEQ ID NO: 11 , 96%, 97%, 98%, 99% or 100% identical amino acid sequences.

In one aspect, isolated polynucleotides encoding bispecific antibodies of the invention are provided.

In one aspect, a host cell comprising the isolated polynucleotide is provided.

In one aspect, there is provided a method of producing a bispecific antibody that binds to CD3/FolR1, comprising the steps of: (a) culturing a host cell under conditions suitable for expressing the bispecific antibody, and optionally ( b) Recover the bispecific antibody.

In one aspect, bispecific antibodies are provided that bind to CD3 and FolR1 produced by the methods described herein above.

In one aspect, a pharmaceutical composition comprising a bispecific antibody of the invention and a pharmaceutically acceptable carrier is provided.

In one aspect, a bispecific antibody of the invention or a pharmaceutical composition of the invention for use as a medicament is provided.

In one aspect, a bispecific antibody of the invention or a pharmaceutical composition of the invention for use in the treatment of cancer is provided.

In one aspect, use of a bispecific antibody of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament is provided.

In one aspect, use of a bispecific antibody of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of cancer is provided.

In one aspect, there is provided a method of treating a disease in a subject comprising administering to the subject an effective amount of a bispecific antibody of the present invention or a pharmaceutical composition of the present invention.

In one aspect, the disease is cancer.

i. definition

Unless otherwise defined below, the terms used herein are those of ordinary usage in the technical field.

The terms "first," "second," or "third," as used herein with respect to antigen binding domains, etc., are used to facilitate distinguishing between each type of moiety when there is more than one. The use of these terms is not intended to confer a specific order or orientation to the parts unless explicitly stated.

The terms "anti-CD3 antibody" and "antibody that binds CD3" refer to an antibody capable of binding CD3 with sufficient affinity to render the antibody useful as a diagnostic and/or therapeutic agent targeting CD3. In one aspect, the anti-CD3 antibody binds to an unrelated, non-CD3 protein to less than about 10% of the binding of the antibody to CD3, as measured by surface plasmon resonance (SPR), for example. In certain aspects, the CD3-binding antibody has a dissociation constant (K _D ) of ≤ 1 μM, ≤ 500 nM, ≤ 200 nM, or ≤ 100 nM. An antibody is said to "specifically bind" CD3 when it has a _KD of 1 μM or less, as measured, for example, by SPR. In certain aspects, the anti-CD3 antibody binds to an epitope of CD3 that is conserved among different species of CD3.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired Antigen-binding activity is sufficient.

An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabodies, linear antibodies, single-chain antibody molecules (eg, scFv and scFab), single-domain antibodies, and Multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, ie the individual antibodies contained in the population are identical and/or bind the same epitope, but Excluded are, for example, antibodies that contain naturally occurring mutations or possible variant antibodies that arise during the production of monoclonal antibody preparations, which are usually present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different epitopes (epitopes), each monoclonal antibody system of a monoclonal antibody preparation is directed against a single epitope on an antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies can be made by a variety of techniques including, but not limited to, fusionoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals comprising all or part of the human immunoglobulin loci , these methods and other exemplary methods for making monoclonal antibodies are described herein.

An "isolated" antibody is one that has been isolated from components of its natural environment. In some aspects, the antibody is purified to greater than 95% or 99% purity, such as by, eg, electrophoresis (eg, SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (eg, ion exchange or reverse phase HPLC) , affinity chromatography, particle size sieve chromatography) method. For a review of methods for assessing antibody purity, see, eg, Flatman et al., J. Chromatogr. B 848:79-87 (2007). In certain aspects, the antibodies provided herein are isolated antibodies.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, and the remainder of the heavy and/or light chain is derived from a different source or species.

A "humanized" antibody system refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will include substantially all of at least one, and usually two variable domains, wherein all or substantially all CDRs correspond to those of the non-human antibody, and all or substantially all FRs Corresponds to those for human antibodies. These variable domains are referred to herein as "humanized variable regions." A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody from which the CDR residues are derived), eg, to restore or improve antibody specificity or affinity. An antibody, such as a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.

A "human antibody" is an antibody having an amino acid sequence corresponding to that produced by a human or human cell or from a non-human source utilizing human antibody repertoire or other human antibody coding sequences The amino acid sequence of the derived antibody. The definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. In certain aspects, the human antibody system is derived from a non-human transgenic mammal, such as a mouse, rat or rabbit. In certain aspects, the human antibody is derived from a fusion tumor cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered herein as human antibodies or human antibody fragments.

The term "antigen-binding domain" refers to that portion of an antibody that comprises a region that binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided, for example, by one or more antibody variable domains (also known as antibody variable regions). In a preferred aspect, the antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in antibody-antigen binding. The variable domains of the heavy and light chains (VH and VL, respectively) of native antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, eg, Kindt et al., Kuby Immunology , 6th ed., WH Freeman & Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, VH or VL domains can be used to separate antibodies that bind a particular antigen from antibodies that bind antigen to screen repertoires of complementary VL or VH domains, respectively. See, eg, Portolano et al, J. Immunol. 150 :880-887 (1993); Clarkson et al, Nature 352:624-628 (1991). "Kabat numbering" as used herein in connection with variable region sequences refers to those described by Kabat et al., Sequences of Proteins of Immunological Interest , 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) numbering system.

As used herein, the amino acid positions of all constant regions and domains of heavy and light chains are as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD The Kabat numbering system in (1991), referred to herein as "numbering according to Kabat numbering" or "Kabat numbering". In particular, the Kabat numbering system (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) at pp. 647-660) is used for kappa and The light chain constant domains CL of the lambda isotype and the Kabat and EU index numbering systems (see pp. 661-723) are used for the heavy chain constant domains (CH1, hinge, CH2 and CH3), which in this case are referred to herein by Refer to "According to the Kabat EU Index Number" or "Kabat EU Index Number" for further clarification.

As used herein, the term "hypervariable region" or "HVR" refers to the various regions in the variable domain of an antibody that are hypervariable in sequence and determine antigen-binding specificity, eg, "complementarity determining regions"("CDRs"). Typically, an antibody contains six CDRs: three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3). Herein, exemplary CDRs include: (a) hypervariable loops present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 ( H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)); (b) CDRs, which are present in amino acids at residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al ., Sequences of Proteins of Immunological Interest , 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and (c) antigen contacts, which are present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al . J. Mol. Biol. 262: 732- 745 (1996)).

Unless otherwise stated, CDRs were determined according to the method described by Kabat et al., supra. Those skilled in the art will understand that the CDR names were determined by Chothia in the aforementioned literature, by the method described by McCallum in the aforementioned literature, or by any other scientifically accepted nomenclature system.

"Framework" or "FR" refers to variable domain residues outside the complementarity determining regions (CDRs). The FRs of the variable domains generally consist of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the HVR and FR sequences usually appear in the VH (or VL) in the following order: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

Unless otherwise specified, CDR residues and other residues (eg, FR residues) in variable domains are The method number described by Kabat et al., supra.

For the purposes of this document, an "acceptor human framework" comprises a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human common framework The framework of the amino acid sequence is defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human common framework may contain the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less less or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

A "human common framework" is a framework that represents the most common amino acid residues in a series of human immunoglobulin VL or VH framework sequences. Typically, human immunoglobulin VL or VH sequences are selected from a subset of variable domain sequences. Typically, the subgroup of sequences is as in Kabat et al., Sequences of Proteins of Immunological Interest , 5th edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3 .

The term "immunoglobulin molecule" as used herein refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also known as the heavy chain variable domain or heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3), also known as the heavy chain variable domain. chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also known as a light chain variable domain or light chain variable region, followed by a light chain constant (CL) domain, also known as light chain constant region. The heavy chains of immunoglobulins can be classified into one of five types, called alpha (IgA), delta (IgD), epsilon (IgE), gamma (IgG) or μ (IgM), some of which can be further divided into are subtypes such as γ ₁ (IgG ₁ ), γ ₂ (IgG ₂ ), γ ₃ (IgG ₃ ), γ ₄ (IgG ₄ ), α ₁ (IgA ₁ ), and α ₂ (IgA ₂ ). Based on the amino acid sequences of their constant domains, immunoglobulin light chains can be classified into one of two types, called kappa (κ) and lambda (λ). An immunoglobulin consists essentially of two Fab molecules and an Fc domain linked by an immunoglobulin hinge region.

The "class" of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some _of these can be further divided into subclasses (isotypes), such as _IgGi , IgG2, _IgG3 , _IgG4 , IgA ₁ and IgA ₂ . The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.

"Fab molecule" refers to a protein consisting of the VH and CH1 domains of the heavy chain ("Fab heavy chain") of an immunoglobulin and the VL and CL domains of the light chain ("Fab light chain").

"Crossover" Fab molecule (also known as "Crossfab") means a Fab molecule in which the variable or constant domains of the Fab heavy and Fab light chains are exchanged (ie, replaced with each other), ie, crossover The Fab molecule comprises a peptide chain (VL-CH1, in the N-terminal to C-terminal direction) consisting of a light chain variable domain VL and a heavy chain constant domain 1 CH1, and a heavy chain variable domain VH and a light chain constant domain A peptide chain composed of CL (VH-CL, in the N-terminal to C-terminal direction). For clarity, in a crossover Fab molecule in which the variable domains of the Fab light chain and Fab heavy chain are swapped, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the "heavy" of the (crossover) Fab molecule. chain". Conversely, in a cross-type Fab molecule in which the constant domains of the Fab light chain and Fab heavy chain are swapped, the peptide chain comprising the variable domain VH of the heavy chain is referred to herein as the "heavy chain" of the (cross-type) Fab molecule.

In contrast, a "conventional" Fab molecule means its native form (ie comprising a heavy chain (VH-CH1, in the N-terminal to C-terminal direction) consisting of heavy chain variable and constant domains and a A Fab molecule of a light chain (VL-CL, in the N-terminal to C-terminal direction) composed of variable and constant domains.

In certain embodiments, the invention relates to bispecific molecules, wherein at least two binding molecules have the same light chain and corresponding remodeled heavy chain, which confer specific binding to respective antigens (eg, CD3 and FolR1). Using this so-called "common light chain" principle, ie combining two or more binders that share a light chain but still have separate specificities, prevents light chain mismatches. Thus, there are fewer by-products during production, which facilitates homogeneous production of bispecific molecules.

The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native sequence Fc regions as well as variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, in particular, one or two amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expressing a particular nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or may include cleavage variants of the full-length heavy chain. This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbered according to the Kabat EU index). Thus, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. Unless otherwise stated, the amino acid sequence of the heavy chain comprising the Fc region (or subunit of the Fc domain as defined herein) is meant herein to be free of the C-terminal glycine-lysine dipeptide. In one aspect, the heavy chain comprising the Fc region (subunit) described herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, according to Kabat) EU index number). In one aspect, the heavy chain comprising an Fc region (subunit) as described herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbered according to the Kabat EU index). Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index), as in Kabat et al ., Sequences of Proteins of Immunological Interest , 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A "subunit" of an Fc domain, as used herein, refers to one of the two polypeptides that form a dimeric Fc domain, ie, a polypeptide comprising a C-terminal constant region capable of stabilizing a self-associating immunoglobulin heavy chain. For example, the subunits of an IgG Fc domain include the IgG CH2 and IgG CH3 constant domains.

"Fusion" means that the components (eg, the Fab molecule and the Fc domain subunit) are linked via peptide bonds, either directly or via one or more peptide linkers.

The term "multispecific" means that an antibody is capable of specifically binding to at least two different epitopes. Multispecific antibodies can be, for example, bispecific antibodies. Typically, bispecific antibodies contain two antigen-binding sites, each of which is specific for a different epitope. In certain aspects, a multispecific (eg, bispecific) antibody is capable of binding two epitopes simultaneously, in particular two epitopes expressed on two different cells.

The term "valent" as used herein refers to the presence of a specified number of antigen-binding sites in an antigen-binding molecule. Thus, the term "monovalent binding to an antigen" refers to the presence of one (and not more than one) antigen-binding sites specific for the antigen in the antigen-binding molecule.

"Antigen binding site" refers to the site, ie, one or more amino acid residues, that provides an antigen binding molecule that interacts with an antigen. For example, the antigen binding site of an antibody comprises amino acid residues from complementarity determining regions (CDRs). Native immunoglobulin molecules usually have two antigen-binding sites, and Fab molecules usually have a single antigen-binding site.

As used herein, the term "antigenic determinant" or "epitope" refers to a site on a polypeptide macromolecule to which an antigen binding domain binds that forms an antigen binding domain-antigen complex (eg, A continuous stretch of amino acids or a conformational configuration consisting of distinct regions of discontinuous amino acids). For example, useful epitopes may be present on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, absent in serum, and/or Present in the extracellular matrix (ECM). In a preferred aspect, the antigen is a human protein.

Unless otherwise specified, "CD3" refers to any native CD3 from any vertebrate source, including mammals, such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys) ) and rodents (eg mice and rats). The term encompasses "full-length", untreated CD3 as well as any form of CD3 that results from treatment in cells. The term also encompasses naturally occurring CD3 variants, eg, splice variants or dual gene variants. In one aspect, the CD3 is human CD3, in particular the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in SEQ ID NO: 112 (without signal peptide). See also UniProt (www.uniprot.org) accession number P07766 (edition 189), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1. In another aspect, the CD3 is cynomolgus (cynomolgus/Macaca fascicularis) CD3, in particular cynomolgus CD3ɛ. The amino acid sequence of cynomolgus CD3ε is shown in SEQ ID NO: 113 (without signal peptide). See also NCBI GenBank No. BAB71849.1. In certain aspects, the antibodies of the invention bind to an epitope of CD3 that is conserved among CD3 antigens from different species, in particular human and cynomolgus CD3. In a preferred aspect, the antibody binds to human CD3.

"Target cell antigen" as used herein refers to an epitope presented on the surface of a target cell, such as a cell in a tumor, such as a cancer cell or a cell of the tumor stroma (in this case a "target cell antigen" tumor cell antigens”). Preferably, the target cell antigen is not CD3, and/or is expressed on a different cell than CD3. In one aspect, the target cell antigen is TYRP-1, in particular human TYRP-1. In another aspect, the target cell antigen is EGFRvIII, in particular human EGFRvIII. In a preferred embodiment, the target antigen is folate receptor 1 (FolR1).

"FolR1" stands for folate receptor 1 (synonyms include but are not limited to folate receptor alpha (FRA), folate binding protein (FBP), MOv18, P15328, FRA1, FRAI), which mediates folate turnover and reduces the entry of folate derivatives into cells Internal protein receptors. The sequence of human FolR1 is shown in SEQ ID NO:137. See also UniProt entry number P15328. Unless otherwise specified, "FolR1" refers to any native FolR1 from any vertebrate source, including mammals, such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys) ) and rodents (eg mice and rats). The term encompasses "full length", untreated FolR1 and any form of FolR1 that results from treatment in a cell. The term also encompasses naturally occurring FolR1 variants, eg, splice variants or dual gene variants. In one aspect, FolR1 is human FolR1.

"TYRP1" or "TYRP-1" stands for tyrosine-related protein 1, which is an enzyme involved in melanin synthesis. The mature form of TYRP1, also originally known as gp75 is a 75 kDa transmembrane glycoprotein. The sequence of human TYRP1 is shown in SEQ ID NO: 114 (without signal peptide). See also UniProt entry number P17643 (version 185). Unless otherwise specified, "TYRP1" refers to any native TYRP1 from any vertebrate source, including mammals, such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys) ) and rodents (eg mice and rats). The term encompasses "full-length", unprocessed TYRP1 as well as any form of TYRP1 that results from processing in a cell. The term also encompasses naturally occurring variants of TYRP1, eg, splice variants or dual gene variants. In one aspect, TYRP1 is human TYRP1.

"EGFRvIII" stands for epidermal growth factor receptor variant III, which is a mutant of EGFR formed by an in-frame deletion of exons 2-7 resulting in a deletion of 267 amino acids and a glycine substitution at the junction . The sequence of human EGFRvIII is shown in SEQ ID NO: 115 (without signal peptide). The sequence of wild-type human EGFR is shown in SEQ ID NO: 116 (without signal peptide). See also UniProt entry number P00533 (version 258). Unless otherwise specified, "EGFRvIII" refers to any native EGFRvIII from any vertebrate source, including mammals, such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys). ) and rodents (eg mice and rats). The term encompasses "full-length", untreated EGFRvIII (but not wild-type EGFR) as well as any form of EGFRvIII that results from treatment in cells (eg, EGFRvIII without a signal peptide). In one aspect, the EGFRvIII is human EGFRvIII.

"Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise stated, "binding affinity" as used herein refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K _D ). Affinity can be measured by well-established methods known in the art, including those described herein. A preferred method for determining affinity is surface plasmon resonance (SPR).

The term "affinity matured" antibody refers to an antibody that has one or more changes in one or more complementarity-determining regions (CDRs) that cause the antibody to respond to an antigen compared to a parent antibody that does not have such changes. improvement in affinity.

"Reduced binding", eg, reduced binding to an Fc receptor, refers to a reduction in the affinity of the respective interaction as measured, eg, by SPR. For clarity, the term also includes reducing the affinity to zero (or below the detection limit of the analytical method), ie the complete abolition of the interaction. In contrast, "increased binding" refers to an increase in the binding affinity of the respective interactions.

"T cell activation" as used herein refers to one or more cellular responses of T lymphocytes (specifically cytotoxic T lymphocytes) selected from the group consisting of: proliferation, differentiation, secretion of cytokines, release of cytotoxic effector molecules, cytotoxicity Activity and expression of activation markers. Suitable assays for measuring T cell activation are known in the art and described herein.

"Modifications that promote the association of the first and second subunits of the Fc domain" are manipulations of the peptide backbone or post-translational modifications to the Fc domain subunits that reduce or prevent a polypeptide comprising an Fc domain subunit from interacting with the Associations of identical polypeptides form homodimers. As used herein, modifications that promote association preferably include separate modifications to each of the two Fc domain subunits (ie, the first and second subunits of the Fc domain) that are desired to associate, wherein These modifications are complementary to each other in order to facilitate the association of the two Fc domain subunits. For example, association-promoting modifications can alter the structure or charge of one or both Fc domain subunits to make them sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between the polypeptide comprising the first Fc domain subunit and the polypeptide comprising the second Fc domain subunit, which are then fused to other groups of each subunit (eg, antigen binding domain). may vary. In some aspects, the modification that promotes the association of the first and second subunits of the Fc domain comprises amino acid mutations, particularly amino acid substitutions, in the Fc domain. In a preferred aspect, the modification that facilitates the association of the first subunit of the Fc domain with the second subunit comprises a separate amino acid mutation, in particular an amine, in each of the two subunits of the Fc domain base acid substitution.

The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion , Immune complex mediated antigen uptake by antigen presenting cells, negative regulation of cell surface receptors (eg, B cell receptors), and B cell activation.

An "activating Fc receptor" is an Fc receptor that, following the engagement of the Fc domain of an antibody, causes a signaling event that stimulates the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRIIIa (CD16a), FcyRI (CD64), FcyRIIa (CD32), and FcyRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism that causes immune effector cells to lyse antibody-coated target cells. A target cell is a cell to which an Fc region-containing antibody or derivative thereof binds, which typically binds specifically to the Fc region via a protein moiety that is N-terminal. As used herein, the term "reduce ADCC" refers to a reduction in the number of target cells lysed in a given time at a given concentration of antibody in the medium surrounding the target cells via the ADCC mechanism defined above, and /or an increase in the concentration of antibody in the medium surrounding the target cells required to achieve lysis of a given number of target cells in a given time via the ADCC mechanism. The reduction in ADCC is mediated relative to the same antibody (but not yet engineered) produced by the same type of host cell using the same standard production, purification, formulation and storage methods (methods known to those of ordinary skill in the art) ADCC. For example, the reduction in ADCC mediated by an antibody comprising an amino acid substitution in the Fc domain that reduces ADCC is relative to ADCC mediated by the same antibody in the Fc domain that does not contain this amino acid substitution. Suitable assays for measuring ADCC are well known in the art (see, eg, PCT Publication No. WO 2006/082515 or PCT Publication No. WO 2012/130831).

The terms "engineering, engineering, engineering" as used herein are taken to include any manipulation of the peptide backbone or post-translational modification of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modification of amino acid sequences, glycation patterns, or side chain groups of individual amino acids, and combinations of these methods.

The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed to obtain the final construct, provided that the final construct has the desired characteristics, eg, decreased binding to Fc receptors or increased association with another peptide. Amino acid sequence deletions and insertions include deletions and insertions of the amino and/or carboxy terminus of amino acids. Preferred amino acid mutations are amino acid substitutions. To alter, for example, the binding characteristics of an Fc region, non-conservative amino acid substitutions, ie, substituting one amino acid for another with different structural and/or chemical properties, are particularly preferred. Amino acid substitutions include non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine) acid, homoserine, 5-hydroxylysine) substitution. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that methods other than genetic engineering, such as chemical modification, to alter the side chain groups of amino acids may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, the substitution of proline at position 329 of the Fc domain to glycine can be represented as 329G, G329, _G329 , P329G or Pro329Gly.

"Percent (%) amino acid sequence identity" described relative to a reference polypeptide sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, when the sequences are aligned and After introducing differences (if necessary), the maximum percent sequence identity is achieved and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished by various means within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR). ) software or FASTA package. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. and its source code is filed with user documentation in the United States Copyright Office, Washington, DC 20559, USA, where it is registered (US Copyright Registration No. TXU510087) and described in WO 2001/007611.

Unless otherwise stated, for the purposes of this article, % amino acid sequence identity values were generated using the ggsearch program of the FASTA suite version 36.3.8c or later to compare matrices with BLOSUM50. The FASTA program suite was developed by W. R. Pearson and D. J. Lipman ("Improved Tools for Biological Sequence Analysis", PNAS 85 (1988) 2444-2448), W. R. Pearson ("Effective protein sequence comparison" Meth. Enzymol. 266 (1996) 227-258), and Pearson et al. (Genomics 46 (1997) 24-36) and available from www.fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or www.ebi.ac.uk/Tools/sss/ fasta is publicly available. Alternatively, use the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) Compare sequences to ensure global rather than local alignments are performed. The percent amino acid identity is provided in the output alignment header.

The term "polynucleotide" or "nucleic acid molecule" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (ie, cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U) ), sugar (ie, deoxyribose or ribose), and phosphate groups. Typically, nucleic acid molecules are described by the sequence of bases, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually represented by 5' to 3'. As used herein, the term nucleic acid molecule includes: deoxyribonucleic acid (DNA), which includes, for example, complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), in particular messenger RNA (mRNA); synthesis of DNA or RNA forms; and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or circular. Additionally, the term nucleic acid molecule includes sense and antisense strands, as well as single- and double-stranded forms. In addition, the nucleic acid molecules described herein may comprise naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate linkages or chemically modified residues. Nucleic acid molecules also include vectors suitable for direct expression of the antibodies of the invention in vitro and/or in vivo, such as in a host or patient. DNA and RNA molecules. Such DNA (eg, cDNA) or RNA (eg, mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, allowing the mRNA to be injected into an individual to generate antibodies in vivo (see, e.g., Stadler (2017) Nature Medicine 23:815-817 or EP 2 101 823 B1).

An "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or in a chromosomal location different from the natural chromosomal location.

"Antibody-encoding isolated polynucleotides (or nucleic acids)" refers to one or more polynucleotide molecules encoding antibody heavy and light chains (or fragments thereof), including such polynucleotides in a single vector or in separate vectors nucleotide molecules, and such polynucleotide molecules present at one or more locations in a host cell.

As used herein, the term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome that has been introduced into the host cell. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

The terms "host cell", "host cell strain" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including progeny cells of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny cells derived therefrom, regardless of the number of passages. The nucleic acid content of the daughter cells may not be exactly the same as the parent cells, but may contain mutations. Screening or selection of mutant progeny cells with the same function or biological activity from the original transformed cells is included herein. Host cells are any type of cellular system that can be used to produce the antibodies of the invention. Host cells include cultured cells, such as cultured mammalian cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or fusionoma cells, yeast cells, insect cells and plant cells, to name a few, but also includes Cells contained in genetically engineered animals, transgenic plants, or cultured plant or animal tissue. In one aspect, the host cells of the present invention are eukaryotic cells, in particular mammalian cells. In one aspect, the host cell is not a cell in the human body.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional components.

"Pharmaceutically acceptable carrier" refers to ingredients in a pharmaceutical composition or formulation other than active ingredients that are not toxic to the individual. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

As used herein, "treatment" (and grammatical variants thereof, such as "in the course of treatment" or "in treatment") refers to a clinical intervention that attempts to alter the natural history of the disease in the subject being treated, and may be prophylactic or clinically pathological. executed during the process. Desired therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease state, and alleviating or improving prognosis. In some aspects, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

A "subject" or "individual" is a mammal. Mammals include, but are not limited to, domestic animals (eg, cattle, sheep, cats, dogs, and horses), primates (eg, humans and non-human primates, such as monkeys), rabbits, and rodents (eg, mice and rats). mouse). In certain aspects, the subject or individual is a human.

A "therapeutically effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective to achieve the desired therapeutic or prophylactic effect at the dose and time period required for administration.

The term "package insert" is used to refer to the instructions usually included in commercial packaging of therapeutic products containing the indications, usage, dosage, route of administration, combination therapy, contraindications and / or warnings, etc. II. COMPOSITIONS AND METHODS

The present invention provides bispecific antibodies that bind CD3 and FolR1. Antibodies exhibit excellent stability combined with other favorable properties for therapeutic applications such as efficacy and safety, pharmacokinetics, and manufacturability. Antibodies of the present invention can be used, for example, to treat diseases such as cancer. A. Bispecific anti- CD3 anti- FolR1 antibody

In one aspect, the invention provides bispecific antibodies that bind to CD3 and FolR1. In one aspect, isolated bispecific antibodies that bind to CD3 and FolR1 are provided. In one aspect, the invention provides bispecific antibodies that specifically bind to CD3 and FolR1. In certain aspects, the bispecific anti-CD3 anti-FolR1 antibody at pH 7.4, 37 relative to the binding activity after 2 weeks at pH 6, -80°C, as determined by surface plasmon resonance (SPR) More than about 90% of the binding activity to CD3 was retained after 2 weeks at °C.

In one aspect, the invention provides a bispecific antibody that binds CD3 and FolR1, wherein the antibody comprises a first antigen binding domain comprising: a heavy chain variable region (VH) comprising SEQ ID NO: 2 heavy chain complementarity determining regions (HCDRs) 1, HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5; and a light chain variable region (VL) comprising the light chain of SEQ ID NO: 8 Complementarity Determining Region (LCDR) 1, LCDR2 of SEQ ID NO:9 and LCDR3 of SEQ ID NO:10.

In one aspect, the antibody is a humanized antibody. In one aspect, the antigen-binding domain is a humanized antigen-binding domain (ie, the antigen-binding domain of a humanized antibody). In one aspect, VH and/or VL are humanized variable regions.

In one aspect, VH and/or VL comprise acceptor human frameworks, such as human immunoglobulin frameworks or human consensus frameworks.

In one aspect, the VH comprises one or more heavy chain framework sequences (ie, FR1, FR2, FR3 and/or FR4 sequences) of the heavy chain variable region sequence of SEQ ID NO: 7. In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 7. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 7. In certain aspects, VH sequences that are at least 95%, 96%, 97%, 98%, or 99% identical contain substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but include The antibody retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO: 7, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO:7. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 7, which includes post-translational modifications of this sequence.

In one aspect, the VL comprises one or more light chain framework sequences (ie, FR1, FR2, FR3 and/or FR4 sequences) of the light chain variable region sequence of SEQ ID NO: 11. In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 11. In certain aspects, VL sequences that are at least 95%, 96%, 97%, 98%, or 99% identical contain substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but include The antibody retains the ability to bind to CD3. In certain aspects, in the amino acid sequence of SEQ ID NO: 11, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). In one aspect, VL comprises the amino acid sequence of SEQ ID NO: 11. Optionally, VL comprises the amino acid sequence of SEQ ID NO: 11, which includes post-translational modifications of this sequence.

In one aspect, VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7, and VL comprises SEQ ID NO: The amino acid sequences of 11 are at least about 95%, 96%, 97%, 98%, or 99% identical to amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO:7 and VL comprises the amino acid sequence of SEQ ID NO:11.

In one aspect, the invention provides an antibody that binds to CD3, wherein the antibody comprises a first antigen binding domain comprising: a VH comprising the amino acid sequence of SEQ ID NO: 7; and VL, which comprises the amino acid sequence of SEQ ID NO: 11.

In another aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1, wherein the antibody comprises a first antigen binding domain comprising: VH comprising SEQ ID NO: 7 sequence; and VL comprising the sequence of SEQ ID NO: 11.

In another aspect, the invention provides a bispecific antibody that binds to CD3 and FolR1, wherein the antibody comprises a first antigen binding domain comprising: a VH comprising SEQ ID NO: 7 heavy chain CDR sequence of VH; and VL comprising the light chain CDR sequence of VL of SEQ ID NO: 11.

In another aspect, the first antigen binding domain comprises the HCDR1, HCDR2 and HCDR3 amino acid sequence of VH of SEQ ID NO:7 and the LCDR1, LCDR2 and LCDR3 amino acid sequence of VL of SEQ ID NO:11.

In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO:7 and is at least 95%, 96%, 97%, 98% or 99% identical in sequence to the framework sequence of the VH of SEQ ID NO:7 sexual framework. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO:7 and a framework having at least 95% sequence identity with the framework sequence of the VH of SEQ ID NO:7. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 7 and a framework having at least 98% sequence identity with the framework sequence of the VH of SEQ ID NO: 7.

In one aspect, the VL comprises the light chain CDR sequence of VL of SEQ ID NO: 11 and is at least 95%, 96%, 97%, 98% or 99% identical in sequence to the framework sequence of VL of SEQ ID NO: 11 sexual framework. In one aspect, the VL comprises the light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 95% sequence identity to the framework sequence of VL of SEQ ID NO: 11. In another aspect, the VL comprises the light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 98% sequence identity with the framework sequence of VL of SEQ ID NO: 11.

In one aspect, the invention provides a bispecific antibody that binds CD3 and FolR1, wherein the antibody comprises a first antigen binding domain comprising a VH sequence as described in any of the aspects provided above and as described above VL sequences described in any aspect provided.

In one aspect, the bispecific antibody comprises human constant regions. In one aspect, bispecific antibodies are immunoglobulin molecules comprising human constant regions, in particular IgG class immunoglobulin molecules comprising human CH1, CH2, CH3 and/or CL domains. Exemplary sequences of human constant domains are given in SEQ ID NOs: 120 and 121 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 122 (human IgG1 heavy chain constant domains CH1-CH2-CH3). In one aspect, the bispecific antibody comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 120 or SEQ ID NO: 121, in particular the amine of SEQ ID NO: 120 An amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence. In one aspect, the bispecific antibody comprises a heavy chain constant region comprising at least about 95%, 96%, 97%, 98%, 99% or the amino acid sequence of SEQ ID NO: 122 100% identical amino acid sequence. In particular, the heavy chain constant region may comprise amino acid mutations in the Fc domain, as described herein.

In one aspect, the first antigen binding domain comprises a human constant region. In one aspect, the first antigen binding moiety is a Fab molecule comprising human constant regions, in particular human CH1 and/or CL domains. In one aspect, the first antigen binding domain comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 120 or SEQ ID NO: 121, in particular SEQ ID NO: 120 An amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence. In particular, the light chain constant region may comprise amino acid mutations as described herein under "Charge Modifications" and/or may comprise one or more (specifically two) N-termini in a crossover Fab molecule Deletion or substitution of amino acids. In some aspects, the first antigen binding domain comprises a heavy chain constant region comprising at least about 95%, 96%, 97%, and 97% of the CH1 domain sequence contained in the amino acid sequence of SEQ ID NO: 122 %, 98%, 99% or 100% identical amino acid sequences. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise amino acid mutations as described herein under "Charge Modifications".

In one aspect, the bispecific antibody is a monoclonal antibody.

In one aspect, the bispecific antibody is an IgG antibody, in particular _an IgG1 antibody. In one aspect, the bispecific antibody is a full-length antibody.

In another aspect, the first and/or second and/or other antigen binding domains are antibody fragments selected from the group consisting of Fv molecules, scFv molecules, Fab molecules and F(ab') ₂ molecules; specific and Fab molecules. In another aspect, the antibody fragment is a diabody, triabody, or tetrabody.

In one aspect, the first antigen binding domain is a Fab molecule. In a preferred aspect, the first antigen binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and CH1 of the Fab light chain and Fab heavy chain, in particular the variable domains VL and VH are replaced with each other (ie, the first antigen binding domain is a crossover Fab molecule).

In another aspect, an antibody according to any of the above aspects may incorporate any of the features, alone or in combination, as described in Section II.A.1.-8. below.

In a preferred aspect, the antibody comprises an Fc domain, in particular an IgG Fc domain, more particularly an IgGl Fc domain. In one aspect, the Fc domain is a human Fc domain. In one aspect, the Fc domain is _a human IgGi Fc domain. An Fc domain consists of first and second subunits, and may incorporate any of the features described below for Fc domain variants (Section II.A.8.), alone or in combination.

In another preferred aspect, the antibody comprises a second antigen-binding domain and optionally a third antigen-binding domain that binds to a second antigen (ie, the antibody is a multispecific antibody, as described further below) (Section II .A.7.). 1. Antibody Fragments

In certain aspects, the antigen binding domains provided herein are antibody fragments.

In one aspect, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab') ₂ molecule, in particular a Fab molecule as described herein. A "Fab'molecule" differs from a Fab molecule by the addition of residues at the carboxy terminus of the CH1 domain, which include one or more cysteines from the hinge region of the antibody. Fab'-SH is a Fab' molecule in which the cysteine residue of the constant domain bears a free thiol group. Pepsin treatment produces an F(ab') ₂ molecule with two antigen binding sites (two Fab molecules) and a portion of the Fc region.

In another aspect, the antibody fragment is a diabody, triabody, or tetrabody. "Diabodies" are antibody fragments that have two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9:129-134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Tri- and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

In another aspect, the antibody fragment is a single chain Fab molecule. A "single-chain Fab molecule" or "scFab" is composed of an antibody heavy chain variable domain (VH), antibody heavy chain constant domain 1 (CH1), antibody light chain variable domain (VL), antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker- VH-CH1, c) VH-CL-Linker-VL-CH1 or d) VL-CH1-Linker-VH-CL. In particular, the linker is a polypeptide having at least 30 amino acids and preferably 32 to 50 amino acids. These single chain Fab molecules are stabilized by natural disulfide bonds between the CL domain and the CH1 domain. In addition, these single chain Fab molecules can be further stabilized by insertion of cysteine residues to generate interchain disulfide bonds (eg, at position 44 of the variable heavy chain and position 100 of the variable light chain according to Kabat numbering) inserted here).

In another aspect, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the variable domains of the heavy chain (VH) and light chain (VL) of an antibody, linked by a linker. In particular, linkers are short polypeptides with 10 to 25 amino acids, and are usually rich in glycine for flexibility and serine or threonine for solubility, and can be The N-terminus of VH is connected to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retained the specificity of the original antibody. For a review of scFv fragments, see, eg, Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, eds. Rosenburg and Moore, (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and US Patent No. 5,571,894 and US Patent No. 5,587,458.

In another aspect, the antibody fragment is a single domain antibody. A "single domain antibody" is an antibody fragment comprising all or part of the heavy chain variable domain of an antibody or all or part of the light chain variable domain of an antibody. In certain aspects, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, eg, U.S. Patent No. 6,248,516 B1).

Antibody fragments can be produced by various techniques including, but not limited to, proteolytic digestion of intact antibodies as described herein and recombinant production in recombinant host cells (eg, E. coli ). 2. Humanized Antibodies

In certain aspects, the antibodies (eg, bispecific antibodies) provided herein are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parental non-human antibody. Typically, a humanized antibody comprises one or more variable domains, wherein the CDRs (or portions thereof) are derived from non-human antibodies and the FRs (or portions thereof) are derived from human antibody sequences. Humanized antibodies will optionally also contain at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody from which the CDR residues are derived), eg, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described in, for example: Riechmann et al., Nature 332:323-329 (1988); Queen et al, Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describes determination region (SDR) transplantation in particular); Padlan, Mol. Immunol. 28:489-498 (1991) (describes "surface remodeling");Dall'Acqua et al., Methods 36:43- 60 (2005) (describes "FR shuffling"); Osbourn et al, Methods 36:61-68 (2005); and Klimka et al, Br. J. Cancer , 83:252-260 (2000) (describes FR Reorganized "guided choice" method).

Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using a "best match" approach (see, eg, Sims et al . J. Immunol. 151:2296 (1993)); derived from light chains or heavy Framework regions of common sequences of human antibodies of a particular subset of chain variable regions (see, e.g.: Carter et al . Proc. Natl. Acad. Sci. USA , 89:4285 (1992); and Presta et al . J. Immunol. , 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, eg, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and derived from screening FR libraries (See, eg, Baca et al., J. Biol. Chem. 272:10678-10684 (1997); and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)). 3. Glycation variants

In certain aspects, the antibodies provided herein (eg, bispecific antibodies) are altered to increase or decrease the degree to which the antibodies undergo glycation. Addition or deletion of glycosylation sites in an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

When the antibody comprises an Fc region, the oligosaccharide to which it is attached can be altered. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, usually N-linked to Asn297 of the CH2 domain of the Fc region. See, eg, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, eg, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose linked to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some aspects, the oligosaccharides in the antibodies of the invention can be modified to generate antibody variants with certain improved properties.

In one aspect, antibody variants are provided that have afucosylated oligosaccharides, ie, oligosaccharide structures lacking (directly or indirectly) fucose linked to the Fc region. These afucosylated oligosaccharides (also known as "defucosylated" oligosaccharides) specifically lack the fucose residue linked to the first GlcNAc in the stem of the biantennary oligosaccharide structure of N-linked oligosaccharides. In one aspect, antibody variants are provided that have an increased proportion of afucosylated oligosaccharides in the Fc region as compared to the native or parent antibody. For example, the proportion of afucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylation is present). oligosaccharides). The percentage of afucosylated oligosaccharides is the sum (average) amount of oligosaccharides lacking fucose residues relative to the sum of all oligosaccharides attached to Asn 297 (e.g. complex, hybrid and high mannose structures) , this percentage is measured via MALDI-TOF mass spectrometry, eg as described in WO 2006/082515. Asn297 refers to the asparagine residue located near position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e. due to the A small sequence change between positions 294 and 300. Such antibodies with an increased proportion of afucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See eg US 2003/0157108; US 2004/0093621.

Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 /0157108; and WO 2004/056312, especially in Example 11); and knockout cell lines, such as CHO cells knocking out the alpha-1,6-fucosyltransferase gene FUT8 (see, eg, Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng ., 94(4):680-688 (2006); and WO 2003/085107); or GDP-fucose Cells with reduced or absent synthesis or transporter activity (see eg US2004259150, US2005031613, US2004132140, US2004110282).

In another aspect, the antibody variant is provided with bisected oligosaccharides, e.g., wherein a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function as described above. Examples of such antibody variants are described, for example, in: Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540 , WO 2003/011878.

Antibody variants having at least one galactose residue on the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087, WO 1998/58964 and WO 1999/22764. 4. Cysteine Engineered Antibody Variants

In certain aspects, it may be desirable to form cysteine-engineered antibodies, such as THIOMAB ^™ antibodies, in which one or more residues of the antibody are replaced with cysteine residues. In preferred aspects, the substituted residues occur at sites accessible to the antibody. By substituting cysteine for these residues, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to bind the antibody to other moieties (eg, drug moieties or linker-drug moieties) bind to form immunoconjugates, as further described herein. Cysteine engineered antibodies can be produced as described, for example, in US Pat. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016040856. 5. Antibody Derivatives

In certain aspects, the antibodies provided herein (bispecific antibodies) can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxolane, poly-1,3,6-tri-oxozan, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohols, and mixtures thereof . Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the amount and/or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in a given condition The therapy below is moderate. 6. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-CD3/anti-FolR1 antibody as described herein, which bind (chemically bind) to one or more therapeutic agents, eg, cytotoxic agents, chemotherapeutic agents, drugs, growth inhibitors, Toxins (eg protein toxins, enzymatically active toxins or fragments thereof of bacterial, fungal, plant or animal origin) or radioisotopes.

In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC), wherein the antibody is conjugated to one or more of the therapeutic agents described above. Linkers are typically used to link antibodies to one or more therapeutic agents. An overview of ADC technology, including examples of therapeutics, drugs, and linkers, is described in Pharmacol Review 68:3-19 (2016).

In another aspect, the immunoconjugate comprises an antibody of the invention bound to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, pokeweed protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, Croton, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin (enomycin) and crescent toxin (tricothecene).

In another aspect, the immunoconjugate comprises an antibody of the invention bound to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include radioisotopes of At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , and Lu. When the radioconjugate is used for detection, it may contain radioactive atoms such as Tc ^99m or I ¹²³ for scintigraphic studies, or for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI). Spin labels such as I ¹²³ , I ¹³¹ , In ¹¹¹ , F ¹⁹ , C ¹³ , N ¹⁵ , O ¹⁷ , gadolinium, manganese or iron.

Conjugates of antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate ( SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminosulfane (IT), iminoester Bifunctional derivatives of (eg, dimethyl adipate HCl), active esters (eg, disuccinimidyl suberic acid), aldehydes (eg, glutaraldehyde), bisazides (eg, bis( p-azidobenzyl)hexamethylenediamine), double nitrogen derivatives (e.g., bis-(p-diazobenzyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate) ) and dual active fluorine compounds (eg, 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described by Vitetta et al. ( Science 238:1098 (1987)). An exemplary chelating agent for binding radionucleotides to antibodies is carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA). See WO 94/11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52 : 127-131 (1992); US Patent No. 5,208,020).

Immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates made with cross-linking agents including, but not limited to, commercially available (eg, from Pierce Biotechnology, Inc. (Rockford, IL., USA). ) commercially available) of BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo- sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimidyl-(4-vinyl sulfo)benzoate). 7. Multispecific Antibodies

The antibodies provided herein are multispecific antibodies, in particular bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different epitopes (eg, two different proteins, or two different epitopes on the same protein). In certain aspects, the multispecific antibody has three or more binding specificities. In certain aspects, one of the binding specificities is for CD3 and the other specificities are for FolR1. In certain aspects, the multispecific antibody can bind to two (or more) different epitopes of CD3. Multispecific (eg, bispecific) antibodies can also be used to localize cytotoxic agents or cells to cells expressing CD3. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and the Knob-in-hole" engineering (see, eg, US Pat. No. 5,731,168, and Atwell et al. J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for the preparation of antibody Fc-heterodimeric molecules (see eg WO 2009/089004); cross-linking two or more antibodies or fragments (see For example, U.S. Patent No. 4,676,980; and Brennan et al., Science , 229: 81 (1985)); use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol. , 148(5): 1547-1553 (1992); and WO 2011/034605); use common light chain technology to circumvent light chain mismatch problems (see e.g. WO 98/50431); use "diabody" technology to prepare bispecific antibody fragments (see e.g. , Hollinger et al., Proc. Natl. Acad. Sci. USA , 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, eg, Gruber et al., J. Immunol. , 152: 5368 (1994)); and triabodies were prepared as described, for example, in Tutt et al . J. Immunol. 147: 60 (1991).

Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies" or DVD-Ig (see, eg, WO 2001/77342 and WO 2008/024715). Further examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010/115589, WO 2010/112193, WO 2010/136172, WO 2010/145792 and WO 2013/026831. Multispecific antibodies or antigen-binding fragments thereof also include "dual-acting FAbs" or "DAFs" that comprise an antigen-binding site that binds CD3 and another different antigen or two different epitopes of CD3 (see e.g. US 2008). /0069820 and WO 2015/095539).

Multispecific antibodies can also be provided asymmetrically, in which domains are crossed in one or more binding arms with the same antigen specificity (so-called "CrossMab" technology), i.e. by exchanging VH/VL domains (see eg WO 2009 /080252 and WO 2015/150447), CH1/CL domains (see eg WO 2009/080253) or complete Fab arms (see eg WO 2009/080251, WO 2016/016299, see also Schaefer et al, PNAS, 108 (2011) ) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20) implementation. Asymmetric Fab arms can also be designed by introducing mutations of charged or uncharged amino acids into the domain interface to direct correct Fab pairing. See eg WO 2016/172485.

Multispecific antibodies are also provided in which binding arms of different specificities share a common light chain. The inventors of the present invention generated bispecific antibodies in which the binding moieties share a common light chain that retains the specificity and efficacy of the parental monospecific antibody for CD3, and can use the same light chain to bind a second antigen (eg, FolR1). The generation of bispecific molecules with common light chains that retain the binding properties of the parent antibody is not straightforward because the common CDRs of hybrid light chains must achieve binding specificities for both targets. In one aspect, the invention provides T cell activating bispecific antigen binding molecules comprising first and second antigen binding moieties, one of which is a Fab molecule capable of specifically binding to CD3, and the other of which is capable of specific binding Fab molecules that bind sexually to FolR1, wherein the first and second Fab molecules have the same VLCL light chain. In one embodiment, the same light chain (VLCL) comprises the light chain CDRs of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In one embodiment, the same light chain (VLCL) comprises SEQ ID NO: 129.

Various other molecular formats for multispecific antibodies are known in the art and are included herein (see, eg, Spiess et al., Mol Immunol 67 (2015) 95-106).

Particular types of multispecific antibodies also included herein are bispecific antibodies designed to bind simultaneously to surface antigens on target cells (eg, tumor cells), such as FolR1, and T cells Activation-invariant components of receptors (TCRs) such as CD3 complexes for retargeting T cells to kill target cells. Thus, in preferred aspects, the antibodies provided herein are multispecific antibodies, in particular bispecific antibodies, wherein one of the binding specificities is directed against CD3 and the other is directed against FolR1.

Examples of bispecific antibody formats useful for this purpose include, but are not limited to, so-called "BiTE (bispecific T cell engager)" molecules, in which two scFv molecules are fused via a flexible linker (see e.g. WO 2004/106381, WO 2005 /061547, WO 2007/042261 and WO 2008/119567; Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and Derivatives thereof, such as tandem diabodies ("TandAb"; Kipriyanov et al, J Mol Biol 293, 41-56 (1999)); "DART" (Dual Affinity Retargeting) molecules, which are based on the diabody format, but Has a C-terminal disulfide bond for additional stabilization (Johnson et al., J Mol Biol 399, 436-449 (2010)), and a so-called triomab, which is a complete mouse/rat IgG hybrid (see Seimetz et al. Human Review: Cancer Treat Rev 36, 458-467 (2010)). Specific T cell bispecific antibody formats included herein are described in WO 2013/026833; WO 2013/026839; WO 2016/020309; and Bacac et al. Oncoimmunology 5(8) (2016) e1203498.

Preferred aspects of the multispecific antibodies of the present invention are described below.

In one aspect, the invention provides an antibody that binds to CD3 comprising a first antigen-binding domain that binds to CD3, as described herein, and a second antigen-binding domain that binds to FolR1 and optionally a third antigen binding domain.

According to a preferred aspect of the present invention, the antigen-binding domain included in the antibody is a Fab molecule (ie, an antigen-binding domain composed of a heavy chain and a light chain, each of which includes a variable domain and a constant domain). In one aspect, the first antigen-binding domain, the second antigen-binding domain, and/or the third antigen-binding domain, when present, are Fab molecules. In one aspect, the Fab molecule is a human Fab molecule. In a preferred aspect, the Fab molecule is a humanized Fab molecule. In yet another aspect, the Fab molecule comprises a human heavy chain constant domain and a human light chain constant domain.

In a preferred aspect according to the invention, the (multispecific) antibody is capable of binding to both the first antigen (ie CD3) and the second antigen (ie FolR1). In one aspect, the (multispecific) antibody is capable of cross-linking T cells and target cells by binding to both CD3 and FolR1. In an even better aspect, the simultaneous binding results in lysis of the target cell, in particular the target cell antigen (ie, FolR1) expressing the tumor cell. In one aspect, the simultaneous binding enables T cell activation. In other aspects, the simultaneous binding results in a cellular response of T lymphocytes, in particular cytotoxic T lymphocytes, selected from the group consisting of: proliferation, differentiation, cytokine secretion, release of cytotoxic effector molecules, cytotoxic activity, and Activation marker performance. In one aspect, binding of the (multispecific) antibody to CD3 without concomitant binding to FolR1 does not activate T cells.

In one aspect, the (multispecific) antibody is capable of redirecting the cytotoxic activity of T cells to target cells. In a preferred aspect, the redirection is independent of MHC-mediated peptide antigen presentation of the target cells and/or T cell specificity.

Preferably, T cells according to any aspect of the invention are cytotoxic T cells. In some aspects, the T cells are CD4 ⁺ or CD8 ⁺ T cells, specifically CD8 ⁺ T cells. a) first antigen binding domain

The (multispecific) antibody of the present invention comprises at least one antigen-binding domain (first antigen-binding domain) that binds to CD3. In a preferred aspect, the CD3 is human CD3 (SEQ ID NO: 112) or cynomolgus monkey CD3 (SEQ ID NO: 113), most particularly human CD3. In one aspect, the first antigen binding domain is cross-reactive (ie, binds specifically to) human and cynomolgus CD3. In some aspects, CD3 is the epsilon subunit of CD3 (CD3 epsilon).

In a preferred aspect, the (bispecific) antibody comprises no more than one antigen binding domain that binds to CD3. In one aspect, the (bispecific) antibody provides a bivalent antibody that binds to CD3.

In one aspect, the antigen binding domain that binds to CD3 is an antibody fragment selected from the group of Fv molecules, scFv molecules, Fab molecules, and F(ab') ₂ molecules. In a preferred aspect, the antigen binding domain that binds to CD3 is a Fab molecule. b) Second antigen binding domain ( and third antigen binding domain )

In certain aspects, the (multispecific) antibodies of the invention comprise at least one antigen binding domain, in particular a Fab molecule, that binds to a second antigen. Preferably the second antigen is not CD3, i.e. is different from CD3. In one aspect, the second antigen is an antigen expressed on cells other than CD3 (eg, expressed on cells other than T cells). In one aspect, the second antigen is a target cell antigen, in particular a tumor cell antigen. In a preferred aspect, the second antigen is FolR1. The second antigen binding domain is capable of directing the (multispecific) antibody to a target site, for example to a specific type of tumor cell expressing the second antigen.

In one aspect, the antigen binding domain that binds to the second antigen (ie, FolR1) is an antibody fragment selected from the group of Fv molecules, scFv molecules, Fab molecules, and F(ab') ₂ molecules. In a preferred aspect, the antigen binding domain that binds to the second antigen is a Fab molecule.

In certain aspects, the (multispecific) antibody comprises two antigen-binding domains, in particular Fab molecules, that bind to a second antigen. In a preferred such aspect, each of the antigen binding domains binds to the same epitope. In a particular aspect, all such antigen binding domains are identical, that is, they have the same molecular format (eg, conventional Fab molecules), and comprise the same amino acid sequence, including the same as described herein of amino acid substitutions in the CH1 and CL domains (if present). In one aspect, the (multispecific) antibody comprises no more than two antigen-binding domains, in particular a Fab molecule that binds to a second antigen.

In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises a human constant region. In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) is a Fab molecule comprising human constant regions, in particular human CH1 and/or CL domains. Exemplary sequences of human constant domains are given in SEQ ID NOs: 120 and 121 (human kappa and lambda CL domains, respectively) and SEQ ID NO: 122 (human IgGl heavy chain constant domains CH1-CH2-CH3). In one aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises a light chain constant region comprising an amine group with SEQ ID NO: 120 or SEQ ID NO: 121 An acid sequence, in particular an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 120. In particular, the light chain constant region may comprise amino acid mutations as described herein under "Charge Modifications" and/or may comprise one or more (specifically two) N-termini in cross-type Fab molecules Deletion or substitution of amino acids. In some aspects, the second antigen-binding domain (and, when present, the third antigen-binding domain) comprises a heavy chain constant region comprising the same as that contained in the amino acid sequence of SEQ ID NO: 122 CH1 domain sequences are at least about 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence. In particular, the heavy chain constant region (specifically the CH1 domain) may comprise amino acid mutations as described herein under "Charge Modifications". TYRP-1

In some aspects of the invention, the second antigen is TYRP-1, specifically human TYRP-1 (SEQ ID NO: 114).

In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) of SEQ ID NO: 15 1. HCDR 2 of SEQ ID NO: 16 and HCDR 3 of SEQ ID NO: 17; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) of SEQ ID NO: 19 1. SEQ ID LCDR2 of NO:20 and LCDR3 of SEQ ID NO:21.

In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) is a humanized antigen-binding domain (ie, the antigen-binding domain of a humanized antibody). In one aspect, the VH and/or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

In one aspect, the VH and/or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human common framework.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises one or more heavy chain framework sequences of SEQ ID NO: 18 (ie, FR1, FR2, FR3 and/or or FR4 sequence). In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 18. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 18. In certain aspects, VH sequences that are at least 95%, 96%, 97%, 98%, or 99% identical contain substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but include The antibody retains the ability to bind to TYRP-1. In certain aspects, in the amino acid sequence of SEQ ID NO: 18, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 18. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 18, which includes post-translational modifications of this sequence.

In one aspect, the VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprises one or more light chain framework sequences of SEQ ID NO: 22 (ie, FR1, FR2, FR3 and/or or FR4 sequence). In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22. In one aspect, VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 22. In one aspect, VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 22. In certain aspects, VL sequences that are at least 95%, 96%, 97%, 98%, or 99% identical contain substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but include The antibody retains the ability to bind to TYRP-1. In certain aspects, in the amino acid sequence of SEQ ID NO: 22, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). In one aspect, VL comprises the amino acid sequence of SEQ ID NO:22. Optionally, VL comprises the amino acid sequence of SEQ ID NO: 22, which includes post-translational modifications of this sequence.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 18 or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and the third antigen binding domain when present) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO:18 and VL comprises the amino acid sequence of SEQ ID NO:22.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises: VH, comprising the sequence of SEQ ID NO: 18; and VL, comprising the sequence of SEQ ID NO: 22 .

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 18 and the VL sequence of SEQ ID NO: 22.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises: VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 18; and VL comprising SEQ ID The light chain CDR sequence of VL of NO: 22.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises the HCDRl, HCDR2 and HCDR3 amino acid sequences of VH of SEQ ID NO: 18 and VL of SEQ ID NO: 22 The amino acid sequences of LCDR1, LCDR2 and LCDR3.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 18 and the framework sequence with the VH of SEQ ID NO: 18 Frameworks with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 18 and a framework having at least 95% sequence identity with the framework sequence of the VH of SEQ ID NO: 18. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 18 and a framework having at least 98% sequence identity with the framework sequence of the VH of SEQ ID NO: 18.

In one aspect, the VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprises the light chain CDR sequence of the VL of SEQ ID NO:22 and the framework sequence of the VL of SEQ ID NO:22 Frameworks with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VL comprises the light chain CDR sequence of VL of SEQ ID NO:22 and a framework having at least 95% sequence identity to the framework sequence of VL of SEQ ID NO:22. In another aspect, VL comprises the light chain CDR sequence of VL of SEQ ID NO:22 and a framework having at least 98% sequence identity to the framework sequence of VL of SEQ ID NO:22. EGFRvIII

In some aspects of the invention, the second antigen is EGFRvIII, in particular human EGFRvIII (SEQ ID NO: 115).

In one aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) of SEQ ID NO: 85 1. HCDR 2 of SEQ ID NO: 86 and HCDR 3 of SEQ ID NO: 87; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) of SEQ ID NO: 89 1. SEQ ID LCDR2 of NO:90 and LCDR3 of SEQ ID NO:91.

In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) is a humanized antigen-binding domain (ie, the antigen-binding domain of a humanized antibody). In one aspect, the VH and/or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

In one aspect, the VH and/or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human common framework.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises one or more heavy chain framework sequences of SEQ ID NO: 88 (ie, FR1, FR2, FR3 and/or or FR4 sequence). In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 88. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 88. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 88. In certain aspects, VH sequences that are at least 95%, 96%, 97%, 98%, or 99% identical contain substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but include The antibody retains the ability to bind to EGFRvIII. In certain aspects, in the amino acid sequence of SEQ ID NO: 88, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO:88. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 88, which includes post-translational modifications of this sequence.

In one aspect, the VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprises one or more light chain framework sequences of SEQ ID NO: 92 (ie, FR1, FR2, FR3 and/or or FR4 sequence). In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 92. In one aspect, VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 92. In one aspect, the VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 92. In certain aspects, VL sequences that are at least 95%, 96%, 97%, 98%, or 99% identical contain substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but include The antibody retains the ability to bind to EGFRvIII. In certain aspects, in the amino acid sequence of SEQ ID NO: 92, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). In one aspect, VL comprises the amino acid sequence of SEQ ID NO:92. Optionally, VL comprises the amino acid sequence of SEQ ID NO: 92, which includes post-translational modifications of this sequence.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 88 or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and the third antigen binding domain when present) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO:88 and VL comprises the amino acid sequence of SEQ ID NO:92.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises: VH, comprising the sequence of SEQ ID NO: 88; and VL, comprising the sequence of SEQ ID NO: 92 .

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 88 and the VL sequence of SEQ ID NO: 92.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises: VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 88; and VL comprising SEQ ID The light chain CDR sequence of VL of NO: 92.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises the HCDRl, HCDR2 and HCDR3 amino acid sequences of VH of SEQ ID NO:88 and VL of SEQ ID NO:92 The amino acid sequences of LCDR1, LCDR2 and LCDR3.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 88 and the framework sequence with the VH of SEQ ID NO: 88 Frameworks with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 88 and a framework having at least 95% sequence identity to the framework sequence of the VH of SEQ ID NO: 88. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 88 and a framework having at least 98% sequence identity with the framework sequence of the VH of SEQ ID NO: 88.

In one aspect, the VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprises the light chain CDR sequence of the VL of SEQ ID NO:92 and the framework sequence with the VL of SEQ ID NO:92 Frameworks with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VL comprises the light chain CDR sequence of VL of SEQ ID NO:92 and a framework having at least 95% sequence identity to the framework sequence of VL of SEQ ID NO:92. In another aspect, the VL comprises the light chain CDR sequence of VL of SEQ ID NO:92 and a framework having at least 98% sequence identity with the framework sequence of VL of SEQ ID NO:92.

In an alternative aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) comprises a VH sequence as in any aspect provided in this section above for EGFRvIII and as in this section above for EGFRvIII VL sequences in any aspect provided in , but based on the following sequences (in listed order) instead of SEQ ID NOs: 85 (HCDR1), 86 (HCDR2), 87 (HCDR3), 88 (VH), 89 (LCDR1 ), 90 (LCDR2), 91 (LCDR3) and 92 (VL): HCDR1 HCDR2 HCDR3 VH LCDR1 LCDR2 LCDR3 VL SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 46 SEQ ID NO: 47 SEQ ID NO: 48 SEQ ID NO: 49 SEQ ID NO: 50 SEQ ID NO: 51 SEQ ID NO: 52 SEQ ID NO: 53 SEQ ID NO: 54 SEQ ID NO: 55 SEQ ID NO: 56 SEQ ID NO: 57 SEQ ID NO: 58 SEQ ID NO: 59 SEQ ID NO: 60 SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 63 SEQ ID NO: 64 SEQ ID NO: 65 SEQ ID NO: 66 SEQ ID NO: 67 SEQ ID NO: 68 SEQ ID NO: 69 SEQ ID NO: 70 SEQ ID NO: 71 SEQ ID NO: 72 SEQ ID NO: 73 SEQ ID NO: 74 SEQ ID NO: 75 SEQ ID NO: 76 SEQ ID NO: 77 SEQ ID NO: 78 SEQ ID NO: 79 SEQ ID NO: 80 SEQ ID NO: 81 SEQ ID NO: 82 SEQ ID NO: 83 SEQ ID NO: 84 SEQ ID NO: 93 SEQ ID NO: 94 SEQ ID NO: 95 SEQ ID NO: 96 SEQ ID NO: 97 SEQ ID NO: 98 SEQ ID NO: 99 SEQ ID NO: 100 SEQ ID NO: 101 SEQ ID NO: 102 SEQ ID NO: 103 SEQ ID NO: 104 SEQ ID NO: 105 SEQ ID NO: 106 SEQ ID NO: 107 SEQ ID NO: 108 FolR1

In some aspects of the invention, the second antigen is FolR1, in particular human FolR1 (SEQ ID NO: 137).

In one aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) of SEQ ID NO: 124 1. HCDR 2 of SEQ ID NO: 125 and HCDR 3 of SEQ ID NO: 126; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) of SEQ ID NO: 8 1. SEQ ID LCDR 2 of NO: 9 and LCDR 3 of SEQ ID NO: 10.

In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) is (derived from) a humanized antibody. In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) is a humanized antigen-binding domain (ie, the antigen-binding domain of a humanized antibody). In one aspect, the VH and/or VL of the second antigen binding domain (and, if present, the third antigen binding domain) are humanized variable regions.

In one aspect, the VH and/or VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprise an acceptor human framework, such as a human immunoglobulin framework or a human common framework.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises one or more heavy chain framework sequences of SEQ ID NO: 123 (ie, FR1, FR2, FR3 and/or or FR4 sequence). In one aspect, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 123. In one aspect, the VH comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 123. In one aspect, the VH comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 123. In certain aspects, VH sequences that are at least 95%, 96%, 97%, 98%, or 99% identical contain substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but include The antibody retains the ability to bind to FolR1. In certain aspects, in the amino acid sequence of SEQ ID NO: 123, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). In one aspect, the VH comprises the amino acid sequence of SEQ ID NO: 123. Optionally, the VH comprises the amino acid sequence of SEQ ID NO: 123, which includes post-translational modifications of this sequence.

In one aspect, the VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprises one or more light chain framework sequences of SEQ ID NO: 11 (ie, FR1, FR2, FR3 and/or or FR4 sequence). In one aspect, the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, VL comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 11. In one aspect, VL comprises an amino acid sequence that is at least about 98% identical to the amino acid sequence of SEQ ID NO: 11. In certain aspects, VL sequences that are at least 95%, 96%, 97%, 98%, or 99% identical contain substitutions (eg, conservative substitutions), insertions or deletions relative to the reference sequence, but include The antibody retains the ability to bind to FolR1. In certain aspects, in the amino acid sequence of SEQ ID NO: 11, a total of 1 to 10 amino acids are substituted, inserted and/or deleted. In certain aspects, substitutions, insertions or deletions occur in regions other than CDRs (ie, in FRs). In one aspect, VL comprises the amino acid sequence of SEQ ID NO: 11. Optionally, VL comprises the amino acid sequence of SEQ ID NO: 11, which includes post-translational modifications of this sequence.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises at least about 95%, 96%, 97%, 98% of the amino acid sequence of SEQ ID NO: 123 or 99% identical amino acid sequence, and the VL of the second antigen binding domain (and the third antigen binding domain when present) comprises at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequences. In one aspect, VH comprises the amino acid sequence of SEQ ID NO: 123 and VL comprises the amino acid sequence of SEQ ID NO: 11.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises: VH, comprising the sequence of SEQ ID NO: 123; and VL, comprising the sequence of SEQ ID NO: 11 .

In another aspect, the second antigen binding domain (and, when present, the third antigen binding domain) comprises the VH sequence of SEQ ID NO: 123 and the VL sequence of SEQ ID NO: 11.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises: VH comprising the heavy chain CDR sequence of VH of SEQ ID NO: 123; and VL comprising SEQ ID The light chain CDR sequence of VL of NO: 11.

In another aspect, the second antigen binding domain (and, if present, the third antigen binding domain) comprises the HCDR1, HCDR2 and HCDR3 amino acid sequences of VH of SEQ ID NO: 123 and VL of SEQ ID NO: 11 The amino acid sequences of LCDR1, LCDR2 and LCDR3.

In one aspect, the VH of the second antigen binding domain (and, if present, the third antigen binding domain) comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 123 and the framework sequence of the VH of SEQ ID NO: 123 Frameworks with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 123 and a framework having at least 95% sequence identity with the framework sequence of the VH of SEQ ID NO: 123. In another aspect, the VH comprises the heavy chain CDR sequence of the VH of SEQ ID NO: 123 and a framework having at least 98% sequence identity with the framework sequence of the VH of SEQ ID NO: 123.

In one aspect, the VL of the second antigen binding domain (and, if present, the third antigen binding domain) comprises the light chain CDR sequence of the VL of SEQ ID NO: 11 and the framework sequence with the VL of SEQ ID NO: 11 Frameworks with at least 95%, 96%, 97%, 98% or 99% sequence identity. In one aspect, the VL comprises the light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 95% sequence identity to the framework sequence of VL of SEQ ID NO: 11. In another aspect, the VL comprises the light chain CDR sequence of VL of SEQ ID NO: 11 and a framework having at least 98% sequence identity with the framework sequence of VL of SEQ ID NO: 11.

In one aspect, the second antigen-binding domain (and, if present, the third antigen-binding domain) comprises the VH sequence as in any aspect provided above in this section and any state as provided above in this section VL sequence in the sample. Anti- TYRP-1 and anti- EGFRvIII antibodies

The invention also provides antibodies that bind to TYRP-1, comprising a VH sequence as provided in any aspect provided in this section above with respect to TYRP-1 and as provided in any aspect provided in this section above with respect to TYRP-1 The VL sequence of (eg, an antibody that binds to TYRP-1 comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) of SEQ ID NO: 15 1, SEQ ID NO: 16 HCDR 2 of SEQ ID NO: 17 and HCDR 3 of SEQ ID NO: 17; and a light chain variable region (VL) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 19, LCDR 2 of SEQ ID NO: 20 and LCDR 3 of SEQ ID NO: 21; or an antibody that binds to TYRP-1, the antibody comprising: VH, comprising the sequence of SEQ ID NO: 18; and VL, comprising the sequence of SEQ ID NO: 22).

The invention also provides antibodies that bind to EGFRvIII, comprising a VH sequence as provided in any aspect of this section above with respect to EGFRvIII and a VL sequence as in any aspect provided in this section above with respect to EGFRvIII (eg, An antibody that binds to EGFRvIII comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 85, HCDR 2 of SEQ ID NO: 86 and SEQ ID NO: HCDR 3 of 87; and a light chain variable region (VL) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 89, LCDR 2 of SEQ ID NO: 90 and LCDR 3 of SEQ ID NO: 91 or an antibody that binds to TYRP-1, the antibody comprising: VH comprising the sequence of SEQ ID NO: 88; and VL comprising the sequence of SEQ ID NO: 92). Anti- FolR1 antibody

The invention also provides antibodies that bind to FolR1 comprising a VH sequence as in any aspect provided in this section above with respect to FolR1 and a VL sequence as in any aspect provided in this section above with respect to FolR1 (eg, An antibody that binds to FolR1, the antibody comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 124, HCDR 2 of SEQ ID NO: 125, and SEQ ID NO: HCDR 3 of 126; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9, and LCDR 3 of SEQ ID NO: 10 or an antibody that binds to FolR1, the antibody comprising: VH comprising the sequence of SEQ ID NO: 123; and VL comprising the sequence of SEQ ID NO: 11).

In another aspect of the invention, an antibody that binds to FolR1 according to any of the above aspects may combine, alone or in combination, any of the features described with respect to an antibody that binds to CD3 (unless the anti-CD3 antibody is clearly specific). properties, such as binding sequences). c) Charge modification

The (bispecific) antibodies of the invention may contain amino acid substitutions in the Fab molecules contained therein, which are particularly effective in reducing mispairing of light chains to mismatched heavy chains (Bence-Jones type by-products), which Ligation may occur in the production of Fab-based multispecific antibodies in which VH/VL exchange occurs in one (or more, if the molecule comprises more than two antigen-binding Fab molecules) of its binding arms (see also PCT publications) No. WO 2015/150447, in particular examples therein, the entire contents of which are incorporated herein by reference). The ratio of desired (bispecific) antibodies to undesired by-products, in particular Bence Jones-type by-products occurring in multispecific antibodies with VH/VL domain exchange in one of its binding arms, can be determined by using Specific amino acid positions in the CH1 and CL domains are improved by introducing oppositely charged amino acids (sometimes referred to herein as "charge modifications").

Thus, in some aspects, the first and second antigen-binding domains of the (bispecific) antibody (and, if present, the third antigen-binding domain) are both Fab molecules, and the In one (specifically the first antigen binding domain), the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, i) in the constant domain CL of the second antigen-binding domain (and, if present, the third antigen-binding domain), the amino acid at position 124 is substituted with a positively charged amino acid (numbering according to Kabat), and wherein In the constant domain CH1 of the second antigen-binding domain (and, if present, the third antigen-binding domain), the amino acid at position 147 or the amino acid at position 213 is replaced by a negatively charged amino acid (according to Kabat). EU Index Number) supersedes; or ii) in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is substituted with a positively charged amino acid (numbering according to Kabat), and wherein in the constant domain CH1 of the first antigen binding domain, The amino acid at position 147 or the amino acid at position 213 is substituted with a negatively charged amino acid (numbering according to the Kabat EU index).

The (bispecific) antibody does not contain the modifications mentioned under i) and ii). The constant domains CL and CH1 of antigen-binding domains with VH/VL exchanged are not replaced with each other (ie remain unexchanged).

In a more specific form, i) In the constant domain CL of the second antigen binding domain (and the third antigen binding domain when present), the amino acid at position 124 is lysine (K), arginine (R) or histamine Acid (H) (according to Kabat numbering) independently substituted, and the amino acid at position 147 or the amine at position 213 in the constant domain CH1 of the second antigen binding domain (and third antigen binding domain when present) The base acid is independently substituted by glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index number); or ii) in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), And in the constant domain CH1 of the first antigen binding domain, the amino acid at position 147 or the amino acid at position 213 is glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index) independent replacement.

In one such aspect, in the constant domain CL of the second antigen binding domain (and, if present, the third antigen binding domain), the amino acid at position 124 is lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) independently substituted and the amino acid at position 147 in the constant domain CH1 of the second antigen binding domain (and third antigen binding domain when present) Or the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In yet another aspect, in the constant domain CL of the second antigen-binding domain (and, if present, the third antigen-binding domain), the amino acid at position 124 is lysine (K), arginine ( R) or histidine (H) (according to Kabat numbering) independently substituted, and in the constant domain CH1 of the second antigen-binding domain (and, when present, the third antigen-binding domain), the amino acid at position 147 is substituted with Glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index number) independently substituted.

In a preferred aspect, in the constant domain CL of the second antigen binding domain (and, if present, the third antigen binding domain), the amino acid at position 124 is lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) independently substituted, and the amino acid at position 123 is lysine (K), arginine (R) or histidine (H) (according to Kabat) numbering) independently substituted, and the amino acid at position 147 is glutamic acid (E) or aspartic acid ( D) (according to the Kabat EU index number) is independently substituted and the amino acid at position 213 is independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index number).

In a more preferred form, the amino acid at position 124 is lysine (K) (according to Kabat numbering) in the constant domain CL of the second antigen-binding domain (and, if present, the third antigen-binding domain). ) and the amino acid at position 123 is substituted with lysine (K) (according to the Kabat numbering), and in the constant domain CH1 of the second antigen binding domain (and, if present, the third antigen binding domain), The amino acid at position 147 was substituted with glutamic acid (E) (numbered according to the Kabat EU index) and the amino acid at position 213 was substituted with glutamic acid (E) (numbered according to the Kabat EU index).

In an even better aspect, in the constant domain CL of the second antigen-binding domain (and, if present, the third antigen-binding domain), the amino acid at position 124 is lysine (K) (according to Kabat). numbering) and the amino acid at position 123 is substituted with arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the second antigen binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (according to the Kabat EU index number) and the amino acid at position 213 was substituted with glutamic acid (E) (according to the Kabat EU index number).

In a preferred aspect, if the amino acid substitution according to the above aspects occurs in the constant domain CL and constant domain CH1 of the second antigen-binding domain (and, if present, the third antigen-binding domain), then the second antigen The constant domain CL of the binding domain (and, if present, the third antigen binding domain) is of the kappa isotype.

Alternatively, amino acid substitutions according to the above aspects may occur in the constant domain CL and constant domain CH1 of the first antigen binding domain, but not the second antigen binding domain (and, where present, the third antigen binding domain) in the constant domain CL and the constant domain CH1. In preferred such aspects, the constant domain CL of the first antigen binding domain is of the kappa isotype.

Thus, in one aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) independently substituted, and in the constant domain CH1 of the first antigen binding domain, the amino acid at position 147 or the amino acid at position 213 is glutamic acid (E) or aspartic acid (D) ( Independently superseded according to the Kabat EU index number).

In another aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is lysine (K), arginine (R) or histidine (H) (according to Kabat). numbering) independently substituted, and in the constant domain CH1 of the first antigen binding domain, the amino acid at position 147 was independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

In yet another aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) independently substituted and the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) and in the first antigen binding domain In the constant domain CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index), and the amino acid at position 213 is substituted with glutamic acid (E) or aspartic acid (D) (numbered according to the Kabat EU index) independently substituted.

In one aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is substituted with lysine (K) (according to Kabat numbering) and the amino acid at position 123 is lysine Acid (K) (according to the Kabat numbering) substitution, and in the constant domain CH1 of the first antigen binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (according to the Kabat EU indexing), and position 213 The amino acid is substituted with glutamic acid (E) (numbered according to the Kabat EU index).

In another aspect, in the constant domain CL of the first antigen binding domain, the amino acid at position 124 is substituted with lysine (K) (according to Kabat numbering) and the amino acid at position 123 is refined Amino acid (R) (numbering according to Kabat) is substituted, and in the constant domain CH1 of the first antigen binding domain, the amino acid at position 147 is substituted with glutamic acid (E) (numbering according to Kabat EU), and position The amino acid at 213 is substituted with glutamic acid (E) (numbered according to the Kabat EU index).

In a preferred aspect, the (bispecific) antibody of the present invention comprises (a) a first antigen-binding moiety that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, wherein the difference between the Fab light chain and the Fab heavy chain is The variable domains VL and VH are substituted for each other and comprise a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2, HCDR 2 of SEQ ID NO: 3 and SEQ ID HCDR 3 of NO: 5, and a light chain variable region (VL) comprising the light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9, and SEQ ID NO: 10 LCDR 3 of the In domain CL, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) (in a preferred form, lysine acid (K) or arginine (R) independently substituted) and the amino acid at position 123 is lysine (K), arginine (R) or histidine (H) (according to Kabat numbering) independently substituted (in a preferred aspect, with lysine (K) or arginine (R)), and in the constant domains of the second antigen binding domain (and, if present, the third antigen binding domain) In CH1, the amino acid at position 147 is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), and the amino acid at position 213 is substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index number) independently substituted. d) Multispecific antibody format

The (bispecific and/or multispecific) antibodies according to the invention may have various configurations. An exemplary configuration is shown in Figure 1 .

In a preferred aspect, the antigen binding domain comprised in the (multispecific) antibody is a Fab molecule. In such aspects, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, etc. may be referred to herein as a first Fab molecule, a second Fab molecule, a third Fab molecule, etc., respectively.

In one aspect, the first antigen binding domain and the second antigen binding domain of the (bispecific) antibody are fused to each other, optionally via a peptide linker. In a preferred aspect, the first antigen binding domain and the second antigen binding domain are each a Fab molecule. In one such aspect, the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain. In another such aspect, the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain. In addition, wherein (i) the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain, or (ii) the second antigen binding domain is C-terminal to the Fab heavy chain In the aspect where the Fab light chain of the first antigen-binding domain and the Fab light chain of the second antigen-binding domain are fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain, the Fab light chain of the first antigen-binding domain and the Fab light chain of the second antigen-binding domain can be fused to each other, optionally via a peptide linker fusion.

(Bispecific) antibodies with a single antigen binding domain (such as a Fab molecule) capable of specifically binding to a second antigen, such as a target cell antigen, such as FolR1 (eg, as in Figure 1A , Figure 1D , Figure 1G , shown in Figure 1H , Figure 1K , Figure 1L ), in particular where internalization of the second antigen is expected following high affinity antigen binding domain binding. In such cases, the presence of more than one antigen binding domain specific for the second antigen may enhance the internalization of the second antigen, thereby reducing its availability.

In other cases, however, there are (bispecific) antibodies (eg, as shown in Figure 1B ) comprising two or more antigen-binding domains (such as Fab molecules) specific for a second antigen, eg, a target cell antigen. , FIG. 1C , FIG. 1E , FIG. 1F , FIG. 1I , FIG. 1J , FIG. 1M , FIG. 1N , FIG. 33A , FIG. 33B ) would be beneficial, for example, to optimize targeting or targeting of target sites Target cell antigen cross-linking.

Thus, in a preferred aspect, the (multispecific, e.g. bispecific) antibody according to the invention comprises a third antigen binding domain.

In one aspect, the third antigen binding domain binds to a second antigen, eg, a target cell antigen, such as FolR1. In one aspect, the third antigen binding domain is a Fab molecule.

In one aspect, the third antigen domain is the same as the second antigen binding domain.

In some aspects, the third antigen binding domain and the second antigen binding domain are each a Fab molecule, and the third antigen binding domain is the same as the second antigen binding domain. Thus, in these aspects, the second antigen binding domain and the third antigen binding domain comprise the same heavy and light chain amino acid sequences and have the same arrangement of domains (ie, conventional or crossover). Furthermore, in these aspects, the third antigen binding domain comprises the same amino acid substitutions (if present) as the second antigen binding domain. For example, amino acid substitutions described herein as "charge modifications" would be made in constant domain CL and constant domain CH1 of each of the second and third antigen-binding domains. Alternatively, these amino acid substitutions can be made in the constant domain CL and constant domain CH1 of the first antigen binding domain (which is also a Fab molecule in the preferred form), but not in the second antigen binding domain and the second antigen binding domain. The constant domain CL and the constant domain CH1 of the three antigen binding domains were performed.

Similar to the second antigen binding domain, the third antigen binding domain is preferably a conventional Fab molecule. All Fab molecules share a common light chain. However, aspects are also envisioned in which the second and third antigen-binding domains are cross-type Fab molecules (and the first antigen-binding domain is a conventional Fab molecule). Therefore, in a preferred aspect, the second antigen binding domain and the third antigen binding domain are each a conventional Fab molecule, and the first antigen binding domain is a cross-type Fab molecule as described herein, that is, in which the Fab heavy chain and light Fab molecules in which the variable domains VH and VL or the constant domains CL and CH1 of the chain are exchanged/replaced with each other. In other aspects, the second antigen binding domain and the third antigen binding domain are each a cross-type Fab molecule, and the first antigen binding domain is a conventional Fab molecule.

If a third antigen binding domain is present, in a preferred aspect, the first antigen binding domain binds to CD3, and the second antigen binding domain and the third antigen binding domain bind to the second antigen (specifically, the target cell) antigen, such as FolR1) binding.

As depicted above and in Figures 33A-E , in one embodiment, a T cell activating bispecific antigen binding molecule comprises at least two Fab fragments with the same light chain (VLCL) and different heavy chains (VHCL), which are Two different antigens confer specificity, ie one Fab fragment is able to specifically bind to the T cell activating antigen CD3 and the other Fab fragment is able to specifically bind to the target cell antigen FolR1.

In a preferred aspect, the (multispecific) antibody of the present invention comprises an Fc domain consisting of a first subunit and a second subunit. The first and second subunits of the Fc domain are capable of stable association.

The (multispecific, eg bispecific) antibodies according to the invention may have different configurations, ie the first antigen binding domain, the second antigen binding domain (and optionally the third antigen binding domain) may be fused to each other and fused to the Fc domain in different ways. The components may be fused directly to each other or preferably via one or more suitable peptide linkers. Where the Fab molecule is fused to the N-terminus of the subunit of the Fc domain, it is usually fused via the immunoglobulin hinge region.

In some aspects, the first antigen binding domain and the second antigen binding domain are each a Fab molecule, and the first antigen binding domain is at the C-terminus of the Fab heavy chain and the N of the first or second subunit of the Fc domain end fusion. In such aspects, the second antigen binding domain can be fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain or the N-terminus of the other of the subunits of the Fc domain. In preferred such aspects, the second antigen binding domain is a conventional Fab molecule, and the first antigen binding domain is a crossover Fab molecule as described herein, ie in which the variable domains VH of the Fab heavy and light chains and Fab molecules in which VL or constant domains CL and CH1 are exchanged/replaced with each other. In other such aspects, the second antigen binding domain is a crossover Fab molecule and the first antigen binding domain is a conventional Fab molecule.

In one aspect, the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and the first antigen-binding domain is at the C-terminus of the Fab heavy chain and the N-terminus of the first subunit or the second subunit of the Fc domain. fused, and the second antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain. In a specific aspect, the (multispecific, eg bispecific) antibody consists essentially of a first Fab molecule and a second Fab molecule, and the Fc domain consists of a first subunit and a second subunit and an optional one or multiple peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is at the C-terminus of the Fab heavy chain and the Fc domain is fused. The N-terminal fusion of the first subunit or the second subunit. Such configurations are schematically depicted in Figures 1G and 1K (in these examples, the first antigen binding domain is a VH/VL cross-type Fab molecule). Optionally, the Fab light chains of the first Fab molecule and the second Fab molecule may additionally be fused to each other.

In another aspect, the first antigen binding domain and the second antigen binding domain are each a Fab molecule, and the first antigen binding domain and the second antigen binding domain are each in the subunit of the C-terminus of the Fab heavy chain and the Fc domain One of the N-terminal fusions. In a specific aspect, the (multispecific, eg bispecific) antibody consists essentially of a first Fab molecule and a second Fab molecule, the Fc domain consisting of a first subunit and a second subunit and optionally One or more peptide linkers consist of a first Fab molecule and a second Fab molecule each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain. 1A and 1D (in these examples, the first antigen binding domain is a VH/VL cross-type Fab molecule, and the second antigen binding domain is a conventional Fab molecule) and in Figure 33E (in these examples, the first Such configurations are schematically depicted for one antigen binding domain and the light chain for the second antigen binding domain. The first Fab molecule and the second Fab molecule can be fused to the Fc domain either directly or via a peptide linker. In a preferred aspect, the first Fab molecule and the second Fab molecule are each fused to an Fc domain via an immunoglobulin hinge region. In a specific aspect, the immunoglobulin hinge region is _a human IgGi hinge region, in particular, wherein the Fc domain is _an IgGi Fc domain.

In some aspects, the first antigen binding domain and the second antigen binding domain are each a Fab molecule, and the second antigen binding domain is at the C-terminus of the Fab heavy chain and the N of the first or second subunit of the Fc domain end fusion. In such aspects, the first antigen-binding domain may be at the C-terminus of the Fab heavy chain and the N-terminus of the Fab heavy chain of the first antigen-binding domain or (as described above) at the N-terminus of the other of the subunits of the Fc domain end fusion. In preferred such aspects, the second antigen-binding domain is a conventional Fab molecule, and the first antigen-binding domain is a cross-type Fab molecule as described herein, ie, wherein the variable domains of the Fab heavy and light chains Fab molecules in which VH and VL or constant domains CL and CH1 are exchanged/replaced with each other. In other such aspects, the second antigen binding domain is a crossover Fab molecule and the first antigen binding domain is a conventional Fab molecule.

In one aspect, the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and the second antigen-binding domain is at the C-terminus of the Fab heavy chain and the N-terminus of the first subunit or the second subunit of the Fc domain. fusion, and the first antigen binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain. In a specific aspect, the (multispecific, eg bispecific) antibody consists essentially of a first Fab molecule and a second Fab molecule, and the Fc domain consists of a first subunit and a second subunit and an optional one or multiple peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is fused at the C-terminus of the Fab heavy chain with the Fc domain. The N-terminal fusion of the first subunit or the second subunit. In Figures 1H and 1L (in these examples, the first antigen binding domain is a VH/VL cross-type Fab molecule, and the second antigen binding domain is a conventional Fab molecule) and in Figures 33C and 33D (in these examples , the light chains of the first and second antigen-binding domains are the same) schematically depicts such a configuration. Optionally, the Fab light chains of the first Fab molecule and the second Fab molecule may additionally be fused to each other.

In a preferred aspect, the first antigen binding domain and the third antigen binding domain are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the second antigen binding domain is fused at the Fab heavy chain. The C-terminus of the heavy chain is fused to the N-terminus of the Fab heavy chain of the first Fab molecule. In a specific aspect, the (multispecific, eg bispecific) antibody consists essentially of a first Fab molecule, a second Fab molecule and a third Fab molecule, the Fc domain consisting of a first subunit and a second subunit and optionally one or more peptide linkers, wherein the second Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab molecule, and the first Fab molecule is between the Fab heavy chain. The C-terminus is fused to the N-terminus of the first subunit of the Fc domain, and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The first Fab molecule and the third Fab molecule can be fused to the Fc domain either directly or via a peptide linker. In a preferred aspect, the first Fab molecule and the third Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In a specific aspect, the immunoglobulin hinge region is _a human IgGi hinge region, in particular, wherein the Fc domain is _an IgGi Fc domain. Optionally, the Fab light chains of the first Fab molecule and the second Fab molecule may additionally be fused to each other.

In another such aspect, the second and third antigen-binding domains are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain, and the first antigen-binding domain is at the Fab heavy chain. The C-terminus of the chain is fused to the N-terminus of the Fab heavy chain of the second antigen binding domain. In a specific aspect, the (multispecific, eg bispecific) antibody consists essentially of a first Fab molecule, a second Fab molecule and a third Fab molecule, the Fc domain consisting of a first subunit and a second subunit and optionally one or more peptide linkers, wherein the first Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second Fab molecule, and the second Fab molecule is between the Fab heavy chain. The C-terminus is fused to the N-terminus of the first subunit of the Fc domain, and wherein the third Fab molecule is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second subunit of the Fc domain. The second Fab molecule and the third Fab molecule can be fused to the Fc domain either directly or via a peptide linker. In a preferred aspect, the second Fab molecule and the third Fab molecule are each fused to the Fc domain via an immunoglobulin hinge region. In a specific aspect, the immunoglobulin hinge region is _a human IgGi hinge region, in particular, wherein the Fc domain is _an IgGi Fc domain. Optionally, the Fab light chains of the first Fab molecule and the second Fab molecule may additionally be fused to each other.

In the configuration of a (multispecific) antibody in which the Fab molecule is fused at the C-terminus of the Fab heavy chain via the immunoglobulin hinge region to the N-terminus of each of the subunits of the Fc domain, two Fab molecules, the hinge region and Fc domains essentially form immunoglobulin molecules. In a preferred aspect, the immunoglobulin molecule is an IgG class immunoglobulin. In an even better aspect, the immunoglobulin is an IgG ₁ subclass immunoglobulin. In another aspect, the immunoglobulin is an IgG ₄ subclass immunoglobulin. In another preferred aspect, the immunoglobulin is a human immunoglobulin. In other aspects, the immunoglobulin is a chimeric immunoglobulin or a humanized immunoglobulin. In one aspect, the immunoglobulin comprises a human constant region, in particular a human Fc region.

In some (multispecific) antibodies of the invention, the Fab light chain of the first Fab molecule and the Fab light chain of the second Fab molecule are fused to each other, optionally via a peptide linker. According to the different configurations of the first Fab molecule and the second Fab molecule, the Fab light chain of the first Fab molecule can be fused at its C-terminus with the N-terminus of the Fab light chain of the second Fab molecule, or the Fab light chain of the second Fab molecule can be fused to the N-terminus of the Fab light chain of the second Fab molecule. The chain can be fused at its C-terminus to the N-terminus of the Fab light chain of the first Fab molecule. Fusion of the first Fab molecule to the Fab light chain of the second Fab molecule further reduces the mismatch between the Fab heavy and light chains, and also reduces the number of plastids required to express some (multispecific) antibodies of the invention.

The antigen binding domains can be fused directly to the Fc domain or to each other, or to the Fc or to each other via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, ( _G4S ) _n , ( _SG4 ) _n , ( _G4S ) _n or G4 _{( SG4} ) _n peptide linkers. "N" is usually an integer of 1 to 10, especially 2 to 4. In one aspect, the peptide linker is at least 5 amino acids in length; in one aspect, 5 to 100 amino acids in length; in another aspect, 10 to 50 amino acids in length base acid. In one aspect, the peptide linker is (GxS) _n or ( _GxS ) _nGm , where G=glycine, S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2, or 3) or (x=4, n=2, 3, 4, or 5, and m=0, 1, 2, or 3), in one aspect, x=4 and n=2 or 3, in another aspect, x=4 and n=2. In one aspect, the peptide linker is (G ₄ S) ₂ . A particularly suitable peptide linker for fusing the Fab light chains of the first and second Fab molecules to each other is ( _{G4S)2 .} An exemplary peptide linker suitable for linking the Fab heavy chains of a first Fab fragment and a second Fab fragment comprises the sequences (D)-( _{G4S)2 (} SEQ ID NOs: 118 and 119). Another suitable such linker comprises the sequence ( _{G4S)4 .} Additionally, the linker may comprise (a portion of) an immunoglobulin hinge region. In particular, in cases where the Fab molecule is fused to the N-terminus of the Fc domain subunit, the fusion can be via an immunoglobulin hinge region or a portion thereof with or without an additional peptide linker.

In some aspects, the bispecific antigen binding molecule comprises a common light chain. In one aspect, the invention provides bispecific antigen binding molecules comprising first and second antigen binding moieties, one of which is a Fab molecule capable of specifically binding to CD3 and the other of which is capable of specifically binding to A Fab molecule of FolR1, wherein the first and second Fab molecules have the same VLCL light chain. In one embodiment, the same light chain (VLCL) comprises the light chain CDRs of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In one embodiment, the same light chain (VLCL) comprises SEQ ID NO: 133.

In one embodiment, the present invention provides a T cell activating bispecific antigen binding molecule comprising (i) a first antigen binding moiety which is a Fab molecule capable of specifically binding to CD3 and comprising at least one selected from the group consisting of SEQ The heavy chain complementarity determining region (CDR) of the group consisting of ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 and at least one selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO : light chain CDRs of the group consisting of 10; (ii) a second antigen binding moiety, which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), and which comprises at least one selected from the group consisting of SEQ ID NO: 124, A heavy chain complementarity determining region (CDR) of the group consisting of SEQ ID NO: 125 and SEQ ID NO: 126 and at least one light selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 chain CDRs.

In one such embodiment, the CD3 antigen binding portion comprises heavy chain CDR1 of SEQ ID NO:2, heavy chain CDR2 of SEQ ID NO:3, heavy chain CDR3 of SEQ ID NO:5, light of SEQ ID NO:8 chain CDR1, light chain CDR2 of SEQ ID NO: 9, and light chain CDR3 of SEQ ID NO: 10, and FolR1 antigen-binding portion 5 comprises heavy chain CDR1 of SEQ ID NO: 124, heavy chain CDR2 of SEQ ID NO: 125, Heavy chain CDR3 of SEQ ID NO:126, light chain CDR1 of SEQ ID NO:8, light chain CDR2 of SEQ ID NO:9, and light chain CDR3 of SEQ ID NO:10.

In one embodiment, the present invention provides a T cell activating bispecific antigen binding molecule comprising (i) a first antigen binding moiety, which is a Fab molecule capable of specifically binding to CD3, comprising a Fab molecule comprising SEQ ID NO: A variable heavy chain of the amino acid sequence of 7 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 11. (ii) a second antigen binding moiety, which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 123 and comprising SEQ ID Variable light chain of the amino acid sequence of NO: 11.

In one embodiment, the present invention provides a T cell activating bispecific antigen binding molecule comprising (i) a first antigen binding moiety, which is a Fab molecule capable of specifically binding to CD3, comprising a Fab molecule comprising SEQ ID NO: A variable heavy chain of the amino acid sequence of 7 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 11, (ii) a second antigen binding moiety capable of being specific for folate receptor 1 (FolR1) A sexually bound Fab molecule comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 123 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 11.

In one embodiment, the T cell activating bispecific antigen binding molecule additionally comprises (iii) a third antigen binding moiety (which is a Fab molecule) capable of specifically binding to FolR1. In one such embodiment, the second and third antigen binding moieties capable of specific binding to FolR1 comprise the same heavy chain complementarity determining region (CDR) and light chain CDR sequences. In one such embodiment, the third antigen binding moiety is the same as the second antigen binding moiety.

Accordingly, in one embodiment, the present invention provides a T cell activating bispecific antigen binding molecule comprising (i) a first antigen-binding portion, which is a Fab molecule capable of specifically binding to CD3, and which comprises at least one selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39 The heavy chain complementarity determining region (CDR) and at least one light chain CDR selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34; (ii) a second antigen binding moiety, which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), and which comprises at least one selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 a heavy chain complementarity determining region (CDR) of the group consisting of and at least one light chain CDR selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34. (iii) a third antigen binding moiety which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), and which comprises at least one selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 a heavy chain complementarity determining region (CDR) of the group consisting of and at least one light chain CDR selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 533, SEQ ID NO: 34.

In one such embodiment, the CD3 antigen binding portion comprises heavy chain CDR1 of SEQ ID NO:37, heavy chain CDR2 of SEQ ID NO:38, heavy chain CDR3 of SEQ ID NO:39, light of SEQ ID NO:32 chain CDR1, light chain CDR2 of SEQ ID NO: 33 and light chain CDR3 of SEQ ID NO: 34, and the FolR1 antigen binding portion comprises heavy chain CDR1 of SEQ ID NO: 16, heavy chain CDR2 of SEQ ID NO: 17, SEQ ID NO: 17 Heavy chain CDR3 of ID NO:18, light chain CDR1 of SEQ ID NO:32, light chain CDR2 of SEQ ID NO:33 and light chain CDR3 of SEQ ID NO:34.

In one embodiment, the present invention provides a T cell activating bispecific antigen binding molecule comprising (i) a first antigen-binding moiety, which is a Fab molecule capable of specifically binding to CD3, comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and an amino group comprising SEQ ID NO: 31 Variable light chain of acid sequence. (ii) a second antigen binding moiety, which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and comprising SEQ ID The variable light chain of the amino acid sequence of NO: 31. (iii) a third antigen binding moiety, which is a Fab molecule capable of specifically binding to folate receptor 1 (FolR1), comprising a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 15 and comprising SEQ ID The variable light chain of the amino acid sequence of NO: 31.

Thus, in one embodiment, the present invention relates to bispecific molecules, wherein at least two binding moieties have the same light chain and corresponding remodeling heavy chain, which confer specificity to the T cell activating antigen CD3 and the target cell antigen FolR1, respectively. sexual union. The use of this so-called "common light chain" principle, ie the combination of two binders that share one light chain but still have separate specificities, prevents light chain mismatches. Thus, there are fewer by-products during production, which facilitates the homogeneous production of T cell activating bispecific antigen molecules.

In some embodiments, the T cell activating bispecific antigen binding molecule comprises an Fc domain consisting of a first subunit and a second subunit capable of stable association. Exemplary embodiments of T cell activating bispecific antigen binding molecules comprising an Fc domain are described below.

In one aspect, the invention provides (multispecific, e.g. bispecific) antibodies comprising a) The first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, and comprises: a heavy chain variable region (VH), the VH comprising the heavy chain complementarity determining region (HCDR) of SEQ ID NO: 2 ) 1. HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) of SEQ ID NO: 8 1, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10; b) a second antigen binding domain that binds to a second antigen, specifically a target cell antigen, more specifically FolR1, wherein the second antigen binding domain is a Fab molecule comprising a light chain variable region (VL), The light chain variable region comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10; c) an Fc domain, which consists of a first subunit and a second subunit; in (i) the first antigen binding domain according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen binding domain according to b), and the second antigen binding domain according to b) is fused at the Fab heavy chain The C-terminus of the heavy chain is fused to the N-terminus of one of the subunits of the Fc domain according to c), or (ii) the second antigen binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen binding domain according to a), and the first antigen binding domain according to a) is fused at the Fab heavy chain The C-terminus of the heavy chain is fused to the N-terminus of one of the subunits of the Fc domain according to c).

In a preferred aspect, the present invention provides a (multispecific) antibody comprising a) The first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, and comprises: a heavy chain variable region (VH), the VH comprising the heavy chain complementarity determining region (HCDR) of SEQ ID NO: 2 ) 1. HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) of SEQ ID NO: 8 1, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10; b) a second antigen binding domain and a third antigen binding domain that bind to a second antigen, in particular an antigen, more particularly FolR1, wherein the second antigen binding domain and the third antigen binding domain are each comprising a light chain variable Fab molecule of region (VL), the target cell light chain variable region comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR of SEQ ID NO: 10 3; and c) an Fc domain, which consists of a first subunit and a second subunit; in (i) the first antigen-binding domain according to a) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain according to b), and the second antigen-binding domain according to b) and the The third antigen binding domains of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c), or (ii) the second antigen-binding domain according to b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a), and the first antigen-binding domain according to a) is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain according to a) The third antigen binding domains of b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

In another aspect, the present invention provides a (multispecific) antibody comprising a) The first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab molecule, and comprises: a heavy chain variable region (VH), the VH comprising the heavy chain complementarity determining region (HCDR) of SEQ ID NO: 2 ) 1. HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) of SEQ ID NO: 8 1, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10; b) a second antigen binding domain that binds to a second antigen, specifically a target cell antigen, more specifically FolR1, wherein the second antigen binding domain is a Fab molecule comprising a light chain variable region (VL), The light chain variable region comprises light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10; c) an Fc domain, which consists of a first subunit and a second subunit; in (i) the first antigen binding domain according to a) and the second antigen binding domain according to b) are each fused at the C-terminus of the Fab heavy chain to the N-terminus of one of the subunits of the Fc domain according to c).

According to any of the above aspects, the components of the (multispecific, eg bispecific) antibody (eg Fab molecule, Fc domain) can be fused directly or via various linkers, in particular via those described herein or in the art peptide linkers known in Suitable non-immunogenic peptide linkers include, for example, ( _G4S ) _n , ( _SG4 ) _n , ( _G4S ) _n or G4 _{( SG4} ) _n peptide linkers, where n is typically 1 to 10, Usually an integer from 2 to 4.

In a preferred aspect, the present invention provides a (bispecific) antibody comprising a) The first antigen-binding domain that binds to CD3, wherein the first antigen-binding domain is a Fab, and comprises a heavy chain variable region (VH), the VH comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2 , HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5; b) The second antigen binding domain and the third antigen binding domain that bind to FolR1, wherein the second antigen binding domain and the third antigen binding domain are each a Fab molecule and comprise a heavy chain variable region (VH), the VH comprising SEQ Heavy chain complementarity determining region (HCDR) 1 of ID NO: 124, HCDR 2 of SEQ ID NO: 125 and HCDR 3 of SEQ ID NO: 126; c) an Fc domain consisting of a first subunit and a second subunit; and Wherein the first antigen binding domain, the second antigen binding domain and the third antigen binding domain comprise a light chain variable region (VL), and the VL comprises the light chain complementarity determining region (LCDR) of SEQ ID NO: 8 1, SEQ ID NO : LCDR 2 of 9 and LCDR 3 of SEQ ID NO: 10.

In one of these aspects according to the invention, in the first subunit of the Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the first subunit of the Fc domain In the secondary unit, the tyrosine residue at position 407 is replaced by a valine residue (Y407V), and optionally, the threonine residue at position 366 is replaced by a serine residue (T366S), and The leucine residue at position 368 was replaced by an alanine residue (L368A) (numbering according to the Kabat EU index).

In another aspect according to these aspects of the invention, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced by a cysteine residue (S354C) or at position 356 The glutamic acid residue is replaced by a cysteine residue (E356C) (specifically the serine residue at position 354 is replaced by a cysteine residue), and in the second unit of the Fc domain , the tyrosine residue at position 349 was additionally replaced by a cysteine residue (Y349C) (numbering according to the Kabat EU index).

In yet another aspect according to these aspects of the invention, in each of the first subunit and the second subunit of the Fc domain, the leucine residue at position 234 is replaced with an alanine residue (L234A), the leucine residue at position 235 was replaced by an alanine residue (L235A), and the proline residue at position 329 was replaced by a glycine residue (P329G) (numbering according to the Kabat EU index) .

In yet another aspect according to these aspects of the invention, the Fc domain is _a human IgGi Fc domain.

In a preferred embodiment, the bispecific antibody comprises: a polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 127; comprising A polypeptide having an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 128; and comprising at least about 95%, 96% of the sequence of SEQ ID NO: 129 , 97%, 98% or 99% identical amino acid sequences (specifically three polypeptides). In another preferred embodiment, the (multispecific, eg bispecific) antibody comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 127; a polypeptide comprising the amino acid sequence of SEQ ID NO: 128 and a polypeptide (specifically three polypeptides) comprising the amino acid sequence of SEQ ID NO: 129.

In a preferred aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: comprising at least about 95%, 96%, 97%, 98% or 99% of the sequence of SEQ ID NO: 127 % polypeptides with identical amino acid sequences; polypeptides comprising at least about 95%, 96%, 97%, 98% or 99% identical amino acid sequences with the sequence of SEQ ID NO: 128; and polypeptides comprising amino acid sequences with SEQ ID NO: 128 : 129 polypeptides (specifically three polypeptides) whose sequences are at least about 95%, 96%, 97%, 98%, or 99% identical to amino acid sequences. In a preferred aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 127; comprising the amino acid sequence of SEQ ID NO: 128 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 129 (specifically, three polypeptides).

In a specific aspect, the bispecific antibody comprises: a polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 130; comprising the same amino acid sequence as SEQ ID NO: 130 A polypeptide having an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of ID NO: 128; and comprising at least about 95%, 96%, 97% of the sequence of SEQ ID NO: 129 %, 98% or 99% identical amino acid sequence of polypeptides (specifically three polypeptides). In another embodiment, the bispecific antibody comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 130; a polypeptide comprising the amino acid sequence of SEQ ID NO: 128; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 129 Polypeptides of amino acid sequence (specifically three polypeptides).

In one aspect, the invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: comprising at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 130 A polypeptide of the amino acid sequence of A polypeptide whose sequence is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence (specifically, three polypeptides). In one aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 130; a polypeptide comprising the amino acid sequence of SEQ ID NO: 128 A polypeptide; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 129 (specifically, three polypeptides).

In a specific aspect, the bispecific antibody comprises: a polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 131; comprising the same amino acid sequence as SEQ ID NO: 131 A polypeptide having an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the sequence of ID NO: 132; and comprising at least about 95%, 96%, 97% of the sequence of SEQ ID NO: 129 %, 98% or 99% identical amino acid sequence of polypeptides (specifically three polypeptides). In another embodiment, the bispecific antibody comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 131; a polypeptide comprising the amino acid sequence of SEQ ID NO: 132; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 129 Polypeptides of amino acid sequence (specifically three polypeptides).

In one aspect, the invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: comprising at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 131 A polypeptide comprising the amino acid sequence of SEQ ID NO: 132; a polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 132; and a polypeptide comprising the same amino acid sequence as SEQ ID NO: 129 A polypeptide whose sequence is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence (specifically, three polypeptides). In one aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1-1, comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 131; comprising the amino acid sequence of SEQ ID NO: 132 and a polypeptide comprising the amino acid sequence of SEQ ID NO: 129 (specifically, three polypeptides).

In a specific aspect, the bispecific antibody comprises: a polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 133; comprising the same amino acid sequence as SEQ ID NO: 133 A polypeptide having an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of ID NO: 134; and comprising at least about 95%, 96%, 97% of the sequence of SEQ ID NO: 129 %, 98% or 99% identical amino acid sequence of polypeptides (specifically three polypeptides). In another embodiment, the bispecific antibody comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 133; a polypeptide comprising the amino acid sequence of SEQ ID NO: 134; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 129 Polypeptides of amino acid sequence (specifically three polypeptides).

In one aspect, the invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: comprising at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 133 A polypeptide having the amino acid sequence of A polypeptide whose sequence is at least about 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence (specifically, three polypeptides). In one aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 133; a polypeptide comprising the amino acid sequence of SEQ ID NO: 134 A polypeptide; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 129 (specifically, three polypeptides).

In a specific aspect, the bispecific antibody comprises: a polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 135; comprising the same amino acid sequence as SEQ ID NO: 135 A polypeptide having an amino acid sequence at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of ID NO: 136; and comprising at least about 95%, 96%, 97% of the sequence of SEQ ID NO: 129 %, 98% or 99% identical amino acid sequence of polypeptides (specifically two polypeptides). In another embodiment, the (multispecific, eg, bispecific) antibody comprises: a polypeptide comprising the amino acid sequence of SEQ ID NO: 135; a polypeptide comprising the amino acid sequence of SEQ ID NO: 136; and A polypeptide (specifically two polypeptides) comprising the amino acid sequence of SEQ ID NO: 129.

In one aspect, the invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: comprising at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 135 A polypeptide comprising the amino acid sequence of SEQ ID NO: 136; a polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 136; and a polypeptide comprising the same amino acid sequence as SEQ ID NO: 129 A polypeptide (specifically two polypeptides) whose amino acid sequence is at least about 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence. In one aspect, the present invention provides a bispecific antibody that binds to CD3 and FolR1, comprising: a polypeptide comprising the amino acid sequence of SEQ ID NO: 135; a polypeptide comprising the amino acid sequence of SEQ ID NO: 136 a polypeptide; and a polypeptide (specifically, two polypeptides) comprising the amino acid sequence of SEQ ID NO: 129. e) Fc domain variants

In a preferred aspect, the (multispecific, e.g. bispecific) antibody of the invention comprises an Fc domain consisting of a first subunit and a second subunit.

The Fc domain of a (multispecific, eg, bispecific) antibody consists of a pair of polypeptide chains comprising the heavy chain domain of an immunoglobulin molecule. For example, the Fc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two subunits of the Fc domain are able to stably associate with each other. In one aspect, the (multispecific, eg, bispecific) antibodies of the invention comprise no more than one Fc domain.

In one aspect, the Fc domain of the (multispecific, eg, bispecific) antibody is an IgG Fc domain. In a preferred aspect, the Fc domain is _an IgGi Fc domain. _In another aspect, the Fc domain is an IgG4 Fc domain. _In a more specific aspect, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (numbering according to the Kabat EU index), in particular the amino acid substitution S228P. This amino acid substitution reduces Fab arm exchange of IgG4 antibodies in vivo (see _Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). In another preferred aspect, the Fc domain is a human Fc domain. In an even better aspect, the Fc domain is _a human IgGi Fc domain. An exemplary sequence of _a human IgGi Fc region is given in SEQ ID No: 117. f) Fc domain modifications to promote heterodimerization

Antibodies according to the invention (multispecific, eg bispecific) comprise different antigen binding domains which can be fused to one or the other of the two subunits of the Fc domain, thus the two subunits of the Fc domain typically comprise in two different polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization allows for several possible combinations of the two polypeptides. To improve the yield and purity of (multispecific, eg, bispecific) antibodies in recombinant production, the introduction of modifications in the Fc domain of (multispecific, eg, bispecific) antibodies that promote the desired polypeptide association would be advantageous.

Thus, in a preferred aspect, the Fc domain of a (multispecific, e.g. bispecific) antibody according to the invention comprises a modification that facilitates the association of the first and second subunits of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect, the modification is performed in the CH3 domain of the Fc domain.

There are various methods of modifying the CH3 domain of an Fc domain to enhance heterodimerization, which are well described eg in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/ 147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, in all such methods, the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are engineered in a complementary manner such that each CH3 domain (or a CH3 domain comprising The heavy chain of the and does not form homodimers between the two first CH3 domains or the two second CH3 domains). These different approaches for improving heavy chain heterodimerization are considered to be related to heavy chain-light chain modifications in (multispecific, eg bispecific) antibodies (eg, VH and VL exchange in one binding arm/ substitution, and the introduction of substituents with oppositely charged amino acids in the CH1/CL interface) binding, which reduces heavy/light chain mismatches and Bence Jones-type by-products.

In a specific aspect, the modification that promotes the association of the first and second subunits of the Fc domain is a so-called "knob-hole" modification, which includes a modification of one of the two subunits of the Fc domain. A "knob" modification and a "hole" modification of the other of the two subunits of the Fc domain.

The "peel-and-mortar" technique is described, for example, in: US 5,731,168; US 7,695,936; Ridgway et al, Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("knob") at the interface of the first polypeptide, and a corresponding cavity ("hole") at the interface of the second polypeptide, so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protrusions are constructed by replacing smaller amino acid side chains on the interface of the first polypeptide with larger side chains, such as tyrosine or tryptophan. Complementary cavities of the same or similar size to the protrusion are formed in the interface of the second polypeptide by replacing the larger amino acid side chain with a smaller amino acid side chain (eg, alanine or threonine).

Thus, in a preferred aspect, in the CH3 domain of the first subunit of the Fc domain of a (multispecific, eg, bispecific) antibody, the amino acid residues are replaced by amino groups with larger side chain bulk. acid residue replacement, resulting in a protrusion within the CH3 domain of the first subunit, which can be positioned in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain, Amino acid residues are replaced by amino acid residues with smaller side chain bulk, resulting in a cavity within the CH3 domain of the second subunit, where the protrusion in the CH3 domain of the second subunit can be positioned. intracavity.

Preferably, the amino acid residue with larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W) .

Preferably, the amino acid residue with smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T) and valine (V) .

Protrusions and cavities can be prepared by altering the nucleic acid encoding the polypeptide (eg, by site-specific mutation or by peptide synthesis).

In one specific aspect, in the first subunit ("knob" subunit) of the Fc domain (of the CH3 domain), the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and In the second subunit ("hole" subunit) of the Fc domain (of the CH3 domain), the tyrosine residue at position 407 was replaced by a valine residue (Y407V). In one aspect, in the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced by a serine residue (T366S), and the leucine residue at position 368 is replaced by alanine Residue substitution (L368A) (numbered according to the Kabat EU index).

In yet another aspect, in the first subunit of the Fc domain, the serine residue at position 354 is additionally replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is half-substituted Cystine residue replacement (E356C) (specifically the serine residue at position 354 is replaced by a cysteine residue), and in the second subunit of the Fc domain, the tyrosine at position 349 The residue was additionally replaced by a cysteine residue (Y349C) (numbering according to the Kabat EU index). Introduction of these two cysteine residues results in the formation of a disulfide bond between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a preferred aspect, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (numbered according to the Kabat EU index). ).

In a preferred aspect, the CD3-binding antigen-binding domain is fused (optionally, via a second antigen bound to a second antigen (ie, FolR1) to the first subunit (comprising a "knob" modification) of the Fc domain binding domains, and/or via peptide linkers). Without wishing to be bound by theory, fusion of the CD3-binding antigen-binding domain to the knob-containing subunit of the Fc domain will (further) minimize the production of antibodies comprising two CD3-binding antigen-binding domains (the other of the two knob-containing polypeptides). space collision).

Other techniques for CH3 modification for forced heterodimerization can be envisaged as an alternative to the present invention and are described in eg WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO 2013/096291.

In one aspect, the heterodimerization method described in EP 1870459 is alternatively used. This method is based on the introduction of oppositely charged amino acids at specific amino acid positions at the CH3/CH3 domain interface between two subunits of the Fc domain. A particular aspect of the (multispecific) antibodies of the invention are the amino acid mutations R409D and K370E in one of the two CH3 domains (of the Fc domain); and the amino acid mutations in the other of the CH3 domains of the Fc domain Mutations D399K and E357K (numbered according to the Kabat EU index).

In another aspect, the (multispecific, eg bispecific) antibody of the invention comprises the amino acid mutation T366 in the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain Amino acid mutations in T366S, L368A, Y407V, and amino acid mutations in the CH3 domain of the first subunit of the Fc domain R409D, K370E, and amino acid mutations in the CH3 domain of the second subunit of the Fc domain D399K , E357K (according to the Kabat EU index number).

In another aspect, the (multispecific, eg, bispecific) antibody of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain, and of the second subunit of the Fc domain. Amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain, or amino acid mutations Y349C, T366W in the CH3 domain of the (multispecific, eg bispecific) antibody comprising the first subunit of the Fc domain Amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and amino acid mutations R409D, K370E in the CH3 domain of the first subunit of the Fc domain, and the second of the Fc domain Amino acid mutations D399K, E357K in the CH3 domain of the subunit (all numbered according to the Kabat EU index).

In one aspect, the heterodimerization method described in WO 2013/157953 is alternatively used. In one aspect, the first CH3 domain comprises the amino acid mutation T366K and the second CH3 domain comprises the amino acid mutation L351D (numbered according to the Kabat EU index). In another aspect, the first CH3 domain further comprises the amino acid mutation L351K. In another aspect, the second CH3 domain further comprises an amino acid mutation selected from the group consisting of Y349E, Y349D and L368E (specifically L368E) (numbered according to the Kabat EU index).

In one aspect, the heterodimerization method described in WO 2012/058768 is alternatively used. In one aspect, the first CH3 domain comprises amino acid mutations L351Y, Y407A, and the second CH3 domain comprises amino acid mutations T366A, K409F. In another aspect, the second CH3 domain further comprises an amino acid mutation at position T411, D399, S400, F405, N390 or K392 selected from, for example: a) T411N, T411R, T411Q, T411K, T411D, T411E or T411W; b) D399R, D399W, D399Y or D399K; c) S400E, S400D, S400R or S400K; d) F405I, F405M, F405T, F405S, F405V or F405W; e) N390R, N390K, or N390D K392M, K392R, K392L, K392F or K392E (according to the Kabat EU index number). In another aspect, the first CH3 domain comprises amino acid mutations L351Y, Y407A, and the second CH3 domain comprises amino acid mutations T366V, K409F. In another aspect, the first CH3 domain comprises amino acid mutations Y407A and the second CH3 domain comprises amino acid mutations T366A, K409F. In another aspect, the second CH3 domain further comprises amino acid mutations K392E, T411E, D399R and S400R (numbered according to the Kabat EU index).

In one aspect, the heterodimerization method described in WO 2011/143545 is alternatively used, eg, amines are performed at positions selected from the group consisting of 368 and 409 (numbered according to the Kabat EU index) base acid modification.

In one aspect, the heterodimerization method described in WO 2011/090762, which also uses the "knob-hole" technique described above, is alternatively used. In one aspect, the first CH3 domain comprises amino acid mutation T366W and the second CH3 domain comprises amino acid mutation Y407A. In one aspect, the first CH3 domain comprises amino acid mutation T366Y and the second CH3 domain comprises amino acid mutation Y407T (numbering according to the Kabat EU index).

In one aspect, the (multispecific, eg bispecific) antibody or Fc domain thereof is of the IgG2 subclass, and alternatively the heterodimerization method described in WO _2010/129304 is used.

In an alternative aspect, modifications that promote association of the first and second subunits of the Fc domain include modifications that mediate electrostatic steering, eg, as described in PCT Publication WO 2009/089004. Typically, this method involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues, making homodimer formation electrostatically unfavorable, but heterologous Dimerization is electrostatically favorable. In one such aspect, the first CH3 domain comprises a negatively charged amino acid (eg glutamic acid (E) or aspartic acid (D), specifically K392D or N392D) to the amines of K392 and N392 amino acid substitution, and the second CH3 domain comprises a positively charged amino acid such as lysine (K) or arginine (R), specifically D399K, E356K, D356K or E357K and more specifically D399K and E356K) amino acid substitution to D399, E356, D356 or E357. In another aspect, the first CH3 domain further comprises an amine of a negatively charged amino acid (eg glutamic acid (E) or aspartic acid (D), specifically K409D or R409D) to K409 or R409 base acid substitution. In another aspect, the first CH3 domain further or alternatively comprises a negatively charged amino acid (eg, glutamic acid (E) or aspartic acid (D)) to the amino groups of K439 and/or K370 Acid substitutions (all numbered according to the Kabat EU index).

In yet another aspect, the heterodimerization method described in WO 2007/147901 is alternatively used. In one aspect, the first CH3 domain comprises amino acid mutations K253E, D282K and K322D, and the second CH3 domain comprises amino acid mutations D239K, E240K and K292D (numbered according to the Kabat EU index).

In another aspect, the heterodimerization method described in WO 2007/110205 is alternatively used.

In one aspect, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbered according to the Kabat EU index). g) Fc domain modifications that reduce Fc receptor binding and / or effector function

The Fc domain confers favorable pharmacokinetic properties to the (multispecific) antibody, including a long serum half-life, which contributes to a good accumulation ratio and favorable tissue-to-blood distribution ratio in target tissues. At the same time, however, this may lead to undesired targeting of (multispecific, eg bispecific) antibodies to cells expressing Fc receptors, rather than to the preferred antigen-bearing cells. In addition, co-activation of the Fc receptor signaling pathway may lead to cytokine release, which in combination with T cell activating properties and the long half-life of (multispecific) antibodies results in the release of cytokine receptors after systemic administration. Hyperactivation and serious side effects. Activation of immune cells other than T cells (carrying Fc receptors) may even reduce the efficacy of (multispecific) antibodies due to potential destruction of T cells (eg via NK cells).

Thus, in a preferred aspect, the Fc domain of a (multispecific, eg bispecific) antibody according to the invention exhibits reduced binding affinity for Fc receptors and/or compared to _a native IgGi Fc domain Reduced effector function. In one such aspect, the Fc domain (or a (multispecific) antibody comprising the Fc domain) is associated with _a native IgGi Fc domain (or _an (multispecific, eg, bispecific) antibody), exhibiting less than 50%, specifically less than 20%, more specifically less than 10% and most specifically less than ₅ % binding affinity to Fc receptors, and/or to native IgGi Fc domain (or a (multispecific, eg bispecific) antibody comprising _a native IgGi Fc domain) exhibits less than 50%, specifically less than 20%, more specifically less than 10% and most specifically Less than 5% effect function. In one aspect, the Fc domain domain (or a (multispecific, eg, bispecific) antibody comprising the Fc domain) does not substantially bind to Fc receptors and/or induce effector function. In a preferred aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically FcγRIIIa. In one aspect, the effector function is one or more selected from the group of CDC, ADCC, ADCP, and cytokine secretion. In a preferred aspect, the effector function is ADCC. In _one aspect, the Fc domain domain exhibits substantially similar binding affinity to the neonatal Fc receptor (FcRn) as compared to the native IgGi Fc domain domain. When an Fc domain (or a (multispecific, eg, bispecific) antibody comprising the Fc domain) exhibits greater than about 70%, specifically greater than about 80%, more specifically greater _than about 90% native IgGi Fc Substantially similar binding to FcRn is achieved when the binding affinity of the domain (or (multispecific, eg, bispecific) antibody comprising _an IgGi Fc domain) to FcRn is determined.

In certain aspects, the engineered Fc domain has reduced binding affinity for the Fc receptor and/or reduced effector function compared to the non-engineered Fc domain. In preferred aspects, the Fc domain of the (multispecific, eg, bispecific) antibody comprises one or more amino acid mutations that reduce the binding affinity and/or effector function of the Fc domain to an Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In aspects in which there is more than one amino acid mutation that reduces the binding affinity of the amino acid for the Fc receptor, the combination of such amino acid mutations can reduce the binding affinity of the Fc domain for the Fc receptor by at least 10-fold , at least 20 times or even at least 50 times. In one aspect, an antibody comprising an engineered Fc domain (multispecific, eg, bispecific) exhibits less than 20%, specifically less than 10%, more specifically less than 5% binding affinity to Fc receptors. In a preferred aspect, the Fc receptor is an Fcγ receptor. In some aspects, the Fc receptor is a human Fc receptor. In some aspects, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. Preferably, binding to each of these receptors is reduced. In some aspects, binding affinity to complement components, in particular to C1q, is also reduced. In one aspect, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. When an Fc domain (or a (multispecific, eg, bispecific) antibody comprising the Fc domain) exhibits greater than about 70% of the non-engineered form of the Fc domain (or the (multispecific) When the binding affinity for FcRn of a bispecific (eg bispecific) antibody) is substantially similar to that of FcRn, the binding affinity of the Fc domain for the receptor is maintained. An Fc domain or a (multispecific) antibody of the invention comprising the Fc domain may exhibit such affinities of greater than about 80% and even greater than about 90%. In certain aspects, the Fc domain of a (multispecific, eg, bispecific) antibody is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector functions may include, but are not limited to, one or more of the following: reduced complement-dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cellular Hormone secretion, decreased antigen uptake mediated by immune complexes of antigen presenting cells, decreased binding to NK cells, decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased Direct signaling induces apoptosis, reduces cross-linking of target-binding antibodies, reduces dendritic cell maturation, or reduces T cell activation. In one aspect, the reduced effector function is selected from one or more of the group consisting of reduced CDC, reduced ADCC, reduced ADCP, and reduced cytokine secretion. In a preferred aspect, the reduced effect function is reduced ADCC. In one aspect, the reduced ADCC is less than 20% of the ADCC induced by the non-engineered Fc domain (or a (multispecific, eg, bispecific) antibody comprising a non-engineered Fc domain).

In one aspect, the amino acid mutation that reduces the binding affinity and/or effector function of the Fc domain to the Fc receptor is an amino acid substitution. In one aspect, the Fc domain comprises amino acid substitutions at positions selected from the group consisting of E233, L234, L235, N297, P331 and P329 (numbered according to the Kabat EU index). In a more specific aspect, the Fc domain comprises amino acid substitutions at positions selected from L234, L235 and P329 (numbered according to the Kabat EU index). In some aspects, the Fc domain comprises amino acid substitutions of L234A and L235A (numbered according to the Kabat EU index). In one such aspect, the Fc domain is _an IgGi Fc domain, in particular _a human IgGi Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid is substituted with P329A or P329G, in particular P329G (numbered according to the Kabat EU index). In one aspect, the Fc domain comprises an amino acid substitution at position P329, and another amino acid substitution at a position selected from the group consisting of E233, L234, L235, N297 and P331 (numbered according to the Kabat EU index). In a more specific aspect, the other amino acid substitution is E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a preferred aspect, the Fc domain comprises amino acid substitutions at positions P329, L234 and L235 (numbered according to the Kabat EU index). In a more preferred aspect, the Fc domain comprises amino acid mutations L234A, L235A and P329G ("P329G LALA", "PGLALA" or "LALAPG"). Specifically, in a preferred aspect, each subunit of the Fc domain comprises the amino acid substitutions L234A, L235A and P329G (numbered according to the Kabat Eu index), ie in the first and second subunits of the Fc domain In each of the units, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the leucine residue at position 329 is replaced by an alanine residue. Proline residues were replaced by glycine residues (P329G) (numbering according to the Kabat EU index).

In one such aspect, the Fc domain is _an IgGi Fc domain, in particular _a human IgGi Fc domain. The amino acid-substituted "P329G LALA" combination almost completely abolished Fcγ receptor (and complement) binding of the human IgGi Fc domain, as described in PCT Publication _No. WO 2012/130831, which is incorporated by reference in its entirety . WO 2012/130831 also describes methods for making such mutant Fc domains and methods for determining their properties (eg Fc receptor binding or effector function).

_IgG4 antibodies exhibit reduced binding affinity to Fc receptors and reduced effector function compared to _IgG1 antibodies. Thus, in some aspects, the Fc domain of a ₍ multispecific) antibody of the invention is an IgG4 Fc domain, in particular a human _IgG4 Fc domain. _In one aspect, the IgG4 Fc domain comprises an amino acid substitution at position S228, in particular the amino acid substitution S228P (numbering according to the Kabat EU index). To further reduce its binding affinity to Fc receptors and/or its effector function, in one aspect, the IgG4 Fc domain comprises an amino acid substitution at position _L235 , in particular amino acid substitution L235E (according to Kabat). EU index number). _In another aspect, the IgG4 Fc domain comprises an amino acid substitution at position P329, in particular the amino acid substitution P329G (numbering according to the Kabat EU index). _In a preferred aspect, the IgG4 Fc domain comprises amino acid substitutions at positions S228, L235 and P329, in particular amino acid substitutions S228P, L235E and P329G (numbered according to the Kabat EU index). These IgG4 Fc domain mutants and their _Fcγ receptor binding properties are described in PCT Publication No. WO 2012/130831, which is incorporated herein by reference in its entirety.

In a preferred aspect, the Fc domain that exhibits reduced binding affinity to an Fc receptor and/or reduced effector function compared to a native IgGi Fc domain is _an Fc domain comprising amino acid substitutions L234A, L235A and as appropriate _The human IgGi Fc domain of _P329G present or the human IgG4 Fc domain comprising the amino acid substitutions S228P, L235E and optionally P329G (numbered according to the Kabat EU index).

In certain aspects, N-glycosylation of the Fc domain has been eliminated. In one such aspect, the Fc domain comprises an amino acid mutation at position N297, in particular an amino acid in which aspartate is replaced by alanine (N297A) or by aspartate (N297D) Superseded (numbered according to the Kabat EU index).

In addition to the Fc domains described above and in PCT Publication No. WO 2012/130831, Fc domains with reduced Fc receptor binding and/or effector function also include Fc domain residues 238, 265, 269, 270, One or more of 297, 327 and 329 substituted for them (US Pat. No. 6,737,056) (numbered according to the Kabat EU Index). Such Fc mutants include Fc mutants with two or more substitutions in amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 Substituted with alanine (US Pat. No. 7,332,581).

Variant Fc domains can be prepared via amino acid deletions, substitutions, insertions or modifications using genetic or chemical methods known in the art. Genetic methods can include site-specific mutagenesis of coding DNA sequences, PCR, gene synthesis, and the like. Correct nucleotide changes can be verified, eg, by sequencing.

Binding to Fc receptors can be readily determined via ELISA, or via surface plasmon resonance (SPR) using standard instruments, such as BIAcore instruments (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Alternatively, the binding affinity of an Fc domain or a (multispecific) antibody comprising an Fc domain to an Fc receptor can be assessed using cell lines known to express a particular Fc receptor (eg, human NK cells expressing the FcγIIIa receptor).

The effector function of an Fc domain or an Fc domain-containing (multispecific, eg, bispecific) antibody can be determined via methods known in the art. Examples of in vitro assay methods for assessing ADCC activity of target molecules are described, for example, in: US Patent No. 5,500,362; Hellstrom et al, Proc Natl Acad Sci USA 83, 7059-7063 (1986); and Hellstrom et al, Proc Natl Acad Sci USA 82, 1499-1502 (1985); US Patent No. 5,821,337; Bruggemann et al, J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays can be used (see eg: ACTI ^™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA); and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega , Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of target molecules can be assessed in vivo in animal models such as those disclosed by Clynes et al. in Proc Natl Acad Sci USA 95, 652-656 (1998).

In some aspects, the binding of the Fc domain to complement components is reduced, in particular to C1q. Thus, in some aspects, wherein the Fc domain is engineered to have reduced effector function, the reduced effector function includes reduced CDC. C1q binding assays can be performed to determine whether an Fc domain or an Fc domain-containing (multispecific, eg, bispecific) antibody can bind C1q and thus have CDC activity. See, eg, C1q and C3c binding ELISAs in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, eg, Gazzano-Santoro et al, J Immunol Methods 202, 163 (1996); Cragg et al, Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

FcRn binding and in vivo clearance/half-life assays can also be performed using methods known in the art (see eg Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929). B. Polynucleotides

The present invention further provides an isolated polynucleotide encoding an antibody of the present invention. The isolated polynucleotide can be a single polynucleotide or a plurality of polynucleotides.

Polynucleotides encoding (multispecific, eg, bispecific) antibodies of the invention may be expressed as a single polynucleotide encoding the entire antibody or as multiple (eg, two or more) polynucleotides co-expressed . Co-expressed polypeptides encoded by polynucleotides can associate via, for example, disulfide bonds or other means to form functional antibodies. For example, the light chain portion of an antibody can be encoded by a separate polynucleotide from the antibody portion comprising the antibody heavy chain. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an antibody. In another example, a portion of an antibody comprising one of the two Fc domain subunits and optionally (a portion of) one or more Fab molecules may be combined with the other comprising the other and optionally the two Fc domain subunits The Fab molecule (part of) is encoded by a separate polynucleotide that is part of the antibody. When co-expressed, the Fc domain subunits will associate to form an Fc domain.

In some aspects, the isolated polynucleotide encodes an entire antibody molecule according to the invention, as described herein. In other aspects, the isolated polynucleotide encodes a polypeptide comprised in an antibody according to the invention, as described herein.

In certain aspects, the polynucleotide or nucleic acid is DNA. In other aspects, the polynucleotides of the invention are RNA, eg, in the form of messenger RNA (mRNA). The RNA of the present invention may be single-stranded or double-stranded RNA. C. Recombination method

Antibodies of the invention can be obtained via solid-state peptide synthesis (eg, Merrifield solid-phase synthesis) or recombinant production. In recombinant production, one or more polynucleotides encoding antibodies, eg, as described above, are isolated and inserted into one or more vectors for further colonization and/or expression in host cells. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one aspect, a vector, in particular an expression vector, comprising a polynucleotide of the invention (ie, a single polynucleotide or a plurality of polynucleotides) is provided. Expression vectors comprising the coding sequences of the antibodies and appropriate transcriptional/translational control signals can be constructed using methods known to those of skill in the art. Such methods include in vitro recombination DNA technology, synthetic technology and in vivo recombination/genetic recombination. See, eg, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. (1989) of technology. The expression vector can be a part of a plastid, a virus, or a nucleic acid fragment. Expression vectors include expression cassettes in which a polynucleotide encoding an antibody (ie, a coding region) is operably associated with a promoter and/or other transcriptional or translational control elements for colonization. A "coding region," as used herein, is a portion of a nucleic acid consisting of codons that are translated into amino acids. Although "stop codons" (TAG, TGA or TAA) are not translated into amino acids, they can be considered part of the coding region (if present), but any flanking sequence (e.g. promoter, ribosome binding site, Transcription terminators, introns, 5' and 3' untranslated regions, etc.) are not part of the coding region. Two or more coding regions may be present in a single polynucleotide construct, eg, on a single vector, or in separate polynucleotide constructs, eg, on separate (different) vectors superior. Furthermore, any vector may contain a single coding region, or may contain two or more coding regions, eg, a vector of the present invention may encode one or more polypeptides that are isolated by post-proteolytic translation or co-translation into final protein. Additionally, a vector, polynucleotide or nucleic acid of the invention may encode a heterologous coding region, fused or unfused to a polynucleotide encoding an antibody of the invention or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs (such as secretion signal peptides) or heterologous functional domains. Operably associated refers to the association of a coding region (eg, a polypeptide) of a gene product with one or more regulatory sequences such that the expression of the gene product is under the influence or control of the regulatory sequences. If induction of promoter function results in transcription of the mRNA encoding the desired gene product and the nature of the linker between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product, nor the ability of the DNA template to be transcribed, then Two DNA segments (eg, a polypeptide coding region and a promoter to which it is associated) are "operably associated." Thus, a promoter region will be operably associated with a nucleic acid encoding a polypeptide if the promoter is capable of affecting the transcription of the nucleic acid. A promoter may be a cell-specific promoter that directs only the bulk transcription of DNA in a predetermined cell. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcription termination signals, can be operably associated with polynucleotides to direct cell-specific transcription. Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, cytomegalovirus (eg, direct early promoter, binding to intron A), simian virus 40 (eg, early promoter) and retroviruses (eg, Rous sarcoma virus). Other transcriptional control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone, and rabbit beta-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcriptional control regions include tissue-specific promoters and enhancers, as well as inducible promoters (eg, tetracycline-inducible promoters). Similarly, various translational control elements are known to those of ordinary skill in the art. These include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (specifically internal ribosomal entry sites or IRES, also known as CITE sequences). Expression cassettes may also include other features such as origins of replication and/or chromosomal integration elements such as retroviral long terminal repeats (LTR) or adeno-associated virus (AAV) inverted terminal repeats (ITR).

The polynucleotides and nucleic acid coding regions of the invention can be associated with additional coding regions encoding secretion or signal peptides that direct secretion of the polypeptides encoded by the polynucleotides of the invention. For example, if secretion of an antibody is desired, DNA encoding a signal sequence can be placed upstream of the nucleic acid encoding the antibody or fragment thereof of the invention. According to the signaling hypothesis, proteins secreted by mammalian cells have signal peptides or secretory leader sequences that are cleaved from mature proteins when the growing protein chain is exported through the crude endoplasmic reticulum. One of ordinary skill in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to produce the secreted or "mature" form of the polypeptide. In certain aspects, a natural signal peptide is used , such as an immunoglobulin heavy or light chain signal peptide or a functional derivative of the sequence that retains the ability to direct secretion with which it is operably associated . Alternatively, heterologous mammalian signal peptides or functional derivatives thereof may be used. For example, the wild-type leader sequence can be replaced with the leader sequence of human histoplasminogen activator (TPA) or mouse beta-glucuronidase.

DNA encoding short protein sequences that can be used to facilitate subsequent purification (e.g., histidine tags) or to aid in labeling the antibody can be included within or at the end of the antibody (fragment) encoding polynucleotide.

In another aspect, a host cell comprising a polynucleotide of the invention (ie, a single polynucleotide or a plurality of polynucleotides) is provided. In certain aspects, host cells comprising the vectors of the present invention are provided. The polynucleotide and the vector may combine any of the features described herein with respect to the polynucleotide and vector, respectively, alone or in combination. In one such aspect, the host cell comprises (eg, has been transformed or transfected with) one or more vectors comprising one or more polynucleotides encoding (a portion of) an antibody of the invention. The term "host cell" as used herein refers to any type of cellular system that can be engineered to produce the antibodies or fragments thereof of the invention. Host cells suitable for replication and supporting expression of antibodies are known in the art. These cells can be transfected or transduced with specific expression vectors where appropriate, and large numbers of cells containing the vector can be grown to inoculate large-scale starter cultures to obtain sufficient quantities of antibody for clinical use. Suitable host cells include prokaryotic microorganisms (such as E. coli) or various eukaryotic cells (such as Chinese hamster ovary cells (CHO), insect cells, etc.). For example, polypeptides may be produced in bacteria, in particular without saccharification. After expression, the polypeptides can be separated from the soluble fraction in the bacterial cell paste and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable hosts for colonization or expression of polypeptide-encoding vectors, including fungal and yeast strains whose glycation pathways have been "humanized", resulting in Generation of polypeptides with partially or fully human glycation patterns. See Gerngross, Nat Biotech 22, 1409-1414 (2004); and Li et al, Nat Biotech 24, 210-215 (2006). Suitable host cells for expression (glycation) of polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, specifically for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be used as hosts. See, eg, US Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (described PLANTIBODIES ^™ technology for the production of antibodies in transgenic plants). Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines include: the monkey kidney CV1 line transformed from SV40 (COS-7); the human embryonic kidney line (as described in Graham et al., J Gen Virol 36, 59 (1977) 293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described in Mather, Biol Reprod 23, 243-251 (1980)); monkey kidney cells (CV1); African green Monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); small Murine mammary tumor cells (MMT 060562); TRI cells (as described in Mather et al., Annals NYAcad Sci 383, 44-68 (1982)); MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including dhfr ^- CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216 (1980)); and myeloma cell lines, such as YO, NSO , P3X63 and Sp2/0. For a review of some suitable mammalian host cell lines for protein production see, eg, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (eds. BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003 ). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, as well as cells contained in transgenic animals, transgenic plants, or cultured plant or animal tissue . In one aspect, the host cells are eukaryotic cells, in particular mammalian cells, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphoid cells (eg, Y0, NSO, Sp20 cells) . In one aspect, the host cell is not a cell in the human body.

Standard techniques are known in the art and foreign genes can be expressed in these systems. Cells expressing a polypeptide comprising the heavy or light chain of an antigen binding domain (eg, an antibody) can be engineered to express other antibody chains as well, so that the product of expression is an antibody that has both heavy and light chains.

In one aspect, a method of producing an antibody according to the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding an antibody as described herein under conditions suitable for expression of the antibody, and optionally The antibody is recovered from the host cell (or host cell culture medium).

The components of the (multispecific, e.g. bispecific) antibodies of the invention may generally be fused to each other. (Multispecific, eg bispecific) antibodies can be designed so that their components are fused directly to each other or indirectly via linker sequences. The composition and length of the linker can be determined according to methods known in the art, and its efficacy can be tested. Examples of linker sequences between different components of a (multispecific) antibody are provided herein. If desired, additional sequences may also be included to incorporate a cleavage site to separate the various components of the fusion, such as endopeptidase recognition sequences.

Antibodies prepared according to the methods described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, particle size chromatography analysis and similar methods. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors or antigens can be used to bind the antibodies. For example, for affinity chromatographic purification of the antibodies of the invention, a matrix with protein A or protein G can be used. Antibodies substantially as described in the Examples can be isolated using sequential protein A or G affinity chromatography and particle size sieve chromatography. The purity of an antibody can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. D. to analyze

The antibodies provided herein can be identified, screened or characterized for their physical/chemical properties and/or biological activity using various assays known in the art. 1. Binding analysis

Binding (affinity) of an antibody to an Fc receptor or target antigen can be obtained, for example, via surface plasmon resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) and the receptor or target protein (such as can be obtained by recombinant expression). of them) were measured. Alternatively, antibody binding to different receptors or target antigens can be assessed using cell lines expressing specific receptors or target antigens (eg, via flow cytometry (FACS)). Specific illustrative and exemplary aspects for measuring binding activity to CD3 are described below. The illustrated assay can be easily adapted to measure binding activity to FolR1 by using the FolR1 antigen in place of the CD3 antigen and minor adjustments readily recognized by the skilled artisan.

In one aspect, binding activity to CD3 is determined by SPR as follows: SPR is performed on a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibody (GE Healthcare, #28958325) was immobilized on a Series S Sensor Chip CM5 (GE Healthcare) at a surface density of 4000 - 6000 resonance units (RU) using standard amine coupling chemistry. HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used as running and dilution buffer. CD3 antibody at a concentration of 2 µg/ml (in 20 mM His, 140 mM NaCl, pH 6.0) was injected at a flow rate of 5 µl/min for approximately 60 seconds. The CD3 antigen used was a heterodimer of CD3 delta and CD3 epsilon ectodomains fused to a human Fc domain (see SEQ ID NOs: 28 and 29) with a knob-hole modification and a C-terminal Avi-tag. CD3 antibody was injected at a concentration of 10 μg/ml for 120 seconds, and dissociation was monitored for approximately 120 seconds at a flow rate of 5 μl/min. The wafer surface was regenerated by two consecutive injections of 10 mM glycine pH 2.1 for approximately 60 seconds each. The bulk refractive index difference was corrected by subtracting the blank injection and by subtracting the response obtained from the blank control flow cell. For evaluation, binding reactions were performed 5 seconds after the end of the injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab response (signal (RU) obtained after capture of CD3 antibody on immobilized anti-Fab antibody). The binding activity of CD3 to the antibody after a particular treatment, relative to the binding activity of CD3 to the antibody after different treatments (also known as the relative activity concentration (RAC)), was determined by referring to the binding activity of the antibody sample after a particular treatment to that after different treatments. The binding activity of the corresponding antibody sample was calculated. 2. Activity Analysis

The biological activity of the (multispecific, eg bispecific) antibodies of the invention can be measured by various assays as described in the Examples. Biological activities may include, for example, induction of proliferation of T cells, induction of signaling in T cells, induction of expression of activation markers in T cells, induction of cytokine secretion by T cells, induction of lysis of target cells (eg, tumor cells), and induction of tumor regression and/or improved survival. E. Compositions, Formulations, and Routes of Administration

In another aspect, the present invention provides pharmaceutical compositions comprising any of the antibodies provided herein, eg, for use in any of the following methods of treatment. In one aspect, a pharmaceutical composition comprises an antibody according to the present invention and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises a (multispecific, eg, bispecific) antibody according to the invention and at least one additional therapeutic agent, eg, as described below.

Also provided is a method of producing an antibody of the invention in a form suitable for in vivo administration, the method comprising (a) obtaining an antibody according to the invention, and (b) formulating the antibody with at least one pharmaceutically acceptable carrier to formulate a formulation of the antibody for in vivo administration.

The pharmaceutical composition of the present invention comprises an effective amount of the antibody dissolved or dispersed in a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are generally nontoxic to recipients at the dosages and concentrations employed, that is, do not produce adverse, allergic or other effects when administered to animals (eg, humans). Adverse reactions (where appropriate). In light of the present disclosure, those skilled in the art will recognize methods of preparing pharmaceutical compositions comprising antibodies and, optionally, additional active ingredients, as exemplified in Remington's Pharmaceutical Sciences 18th Edition (Mack Printing Company, 1990), which begins with Incorporated herein by reference. In addition, for animal (eg, human) administration, it should be understood that the formulation should meet the sterility, pyrogenicity, general safety, and purity standards required by FDA's Office of Biologics Standards or authorities in other countries. Preferred compositions are lyophilized formulations or aqueous solutions. "Pharmaceutically acceptable carrier" as used herein includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (eg, antibacterial, antifungal), isotonic agents , absorption delaying agents, salts, preservatives, antioxidants, proteins, drugs, drug stabilizers, polymers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavors, dyes, Such materials and combinations thereof are known to those of ordinary skill in the art (see, eg, Remington's Pharmaceutical Sciences, p. 18th edition, Mack Printing Company, 1990, pp. 1289-1329, which is hereby incorporated by reference). Unless any conventional carrier is incompatible with the active ingredient, it is contemplated for use in pharmaceutical compositions.

The (multispecific, eg, bispecific) antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and if local treatment is desired, then Intralesional administration can be employed. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be via any suitable route, eg, via injection, eg, intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic.

Parenteral compositions include those designed for administration by injection, eg, subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection. For injection, the antibodies of the invention can be formulated in aqueous solutions, in particular in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer . The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the antibody may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use. Sterile injectable solutions are prepared by incorporating the antibody of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated below, as required. Sterility can be readily achieved, for example, by filtration through sterile membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield the active ingredient with any additional desired ingredient from a previously filtered sterile liquid medium of powder. If necessary, the liquid medium should be properly buffered and the liquid diluent made isotonic prior to injection of sufficient saline or dextrose. The composition must be stable under the conditions of manufacture and storage, and must be resistant to contamination by microorganisms such as bacteria and fungi. It should be understood that endotoxin contamination should be minimized at safe concentrations, eg, less than 0.5 ng/mg protein. Suitable pharmaceutically acceptable carriers include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethyl Benzylammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methylparaben or paraben propyl formate; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins Proteins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, histidine, arginine or lysine; monosaccharides, di- Sugars and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes ( such as zinc protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, and the like. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (eg, sesame oil) or synthetic fatty acid esters (eg, ethyl oleate or triglycerides) or liposomes.

The active ingredient can be entrapped, for example, in microcapsules prepared via coacervation techniques or via interfacial polymerization (eg, hydroxymethylcellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal pharmaceuticals In delivery systems such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18th edition, Mack Printing Company, 1990). Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing polypeptides in the form of shaped articles such as films or microcapsules. In particular aspects, delayed absorption of the injectable compositions can be brought about by the use in the compositions of substances which delay absorption, for example, aluminum monostearate, gelatin, or combinations thereof.

In addition to the compositions previously described, antibodies can also be formulated as depot preparations. Such long-acting formulations can be administered via implantation (eg, subcutaneously or intramuscularly) or via intramuscular injection. Thus, for example, antibodies can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in a useful oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical compositions comprising the (multispecific, e.g., bispecific) antibodies of the invention can be manufactured using conventional mixing, dissolving, emulsifying, encapsulating, encapsulating, or lyophilizing methods. The pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or auxiliaries that facilitate processing of proteins into pharmaceutically acceptable preparations. Appropriate formulations will depend on the route of administration chosen.

Antibodies can be formulated into compositions in free acid or base, neutral or salt form. A pharmaceutically acceptable salt is one that retains substantially the biological activity of the free acid or base. These include acid addition salts, such as those formed with free amine groups of protein compositions, or those formed with inorganic acids (eg, hydrochloric or phosphoric acid) or organic acids (such as acetic, oxalic, tartaric, or mandelic acids) Wait. Salts with free carboxyl groups can also be derived from: inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms. F. Therapeutic methods and compositions

Any of the (multispecific, eg, bispecific) antibodies provided herein can be used in methods of therapy. The antibodies of the present invention can be used as immunotherapeutic agents, eg, in the treatment of cancer.

For use in a method of treatment, the (multispecific, eg bispecific) antibodies of the invention will be formulated, administered and administered in a manner consistent with good medical practice. In this case, considerations include the specific condition to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the drug, the method of administration, the schedule of administration, and what is known to the medical practitioner other factors.

In one aspect, a (multispecific, e.g., bispecific) antibody of the invention is provided for use as a medicament. In another aspect, a (multispecific, e.g., bispecific) antibody of the invention is provided for use in the treatment of disease. In certain aspects, (multispecific, e.g., bispecific) antibodies of the invention for use in a method of treatment are provided. In one aspect, the invention provides a (multispecific, e.g., bispecific) antibody of the invention for use in the treatment of a disease in a subject in need thereof. In certain aspects, the invention provides a (multispecific, eg, bispecific) antibody for use in a method of treating a subject having a disease, the method comprising administering to the subject an effective amount of antibodies. In certain aspects, the disease to be treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In certain aspects, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, eg, an anticancer agent (if the disease to be treated is cancer). In another aspect, the present invention provides an antibody for inducing lysis of target cells, in particular tumor cells. In certain aspects, the invention provides a (multispecific, eg, bispecific) antibody of the invention for use in a method of inducing lysis of target cells, in particular tumor cells, in a subject, the The method comprises administering to a subject an effective amount of an antibody to induce lysis of target cells. A "subject" according to any of the above aspects is a mammal, preferably a human.

In another aspect, the invention provides the use of a (multispecific, eg, bispecific) antibody of the invention for the manufacture or preparation of a medicament. In one aspect, the medicament is for treating a disease in a subject in need thereof. In another aspect, the medicament is used in a method of treating a disease, the method comprising administering to a subject suffering from the disease a therapeutically effective amount of the medicament. In certain aspects, the disease to be treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In one aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, eg, an anticancer agent (if the disease to be treated is cancer). In another aspect, the agent is used to induce lysis of target cells, in particular tumor cells. In yet another aspect, an agent is used in a method of inducing lysis of target cells, in particular tumor cells, in an individual, the method comprising administering to the subject an effective amount of the agent to induce lysis of the target cells. A "subject" according to any of the above aspects may be a mammal, preferably a human.

In another aspect, the present invention provides a method of treating a disease. In one aspect, the method comprises administering to a subject suffering from the disease a therapeutically effective amount of an antibody of the invention. In one aspect, a composition in a pharmaceutically acceptable form comprising an antibody of the invention is administered to the subject. In certain aspects, the disease to be treated is a proliferative disorder. In a preferred aspect, the disease is cancer. In certain aspects, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, eg, an anticancer agent (if the disease to be treated is cancer). A "subject" according to any of the above aspects may be a mammal, preferably a human.

In another aspect, the present invention provides a method of inducing lysis of target cells, in particular tumor cells. In one aspect, the method comprises contacting target cells with an antibody of the invention in the presence of T cells, particularly cytotoxic T cells. In another aspect, a method of inducing lysis of target cells, in particular tumor cells, in a subject is provided. In one such aspect, the method comprises administering to the subject an effective amount of an antibody of the invention to induce lysis of target cells. In one aspect, the "subject" is a human.

In certain aspects, the disease to be treated is a proliferative disorder, particularly cancer. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer , gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell cancer, bone cancer and kidney cancer. Other cell proliferative disorders that can be treated using the antibodies of the invention include, but are not limited to, tumors localized in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid) , pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, chest and genitourinary system. Also included are precancerous conditions or lesions and cancer metastases. In certain embodiments, the cancer is selected from the group consisting of kidney cancer, bladder cancer, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer, and prostate cancer. In one aspect, especially when the antibody is a bispecific antibody that binds FolR1 as the second antigen, the cancer is a cancer that expresses (or overexpresses) FolR1. In one aspect, especially when the antibody is a bispecific antibody that binds FolRl as the second antigen, the cancer is ovarian, lung, breast or kidney cancer. The skilled artisan readily recognizes that, in many cases, the antibody may not provide a cure, but only a partial benefit. In some aspects, physiological changes that have some benefit are also considered therapeutically beneficial. Thus, in some aspects, the amount of antibody that provides a physiological change is considered an "effective amount." The individual, patient or subject in need of treatment is typically a mammal, more particularly a human.

In some aspects, an effective amount of an antibody of the invention is administered to a subject to treat a disease.

For the prevention or treatment of disease, the appropriate dose of the antibodies of the invention (either alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's body weight, the type of antibody, the Severity and course of disease, administration of the antibody for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, patient's clinical history and response to the antibody, and the judgment of the attending physician. In any event, the practitioner responsible for administration will determine the concentration of one or more active ingredients in the composition and the appropriate dosage for the individual individual. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administrations, and pulse infusions.

The antibody is suitably administered to the patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 µg/kg to 15 mg/kg (eg, 0.1 mg/kg - 10 mg/kg) of the antibody may be administered, eg, via one or more divided administrations or via continuous infusion. The initial candidate dose administered to the patient. A typical daily dose may range from about 1 µg/kg to 100 mg/kg or more, depending on the factors above. For repeated administrations over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of the antibody will be in the range of about 0.005 mg/kg to about 10 mg/kg. In other non-limiting examples, dosages may also include about 1 μg/kg body weight, about 5 μg/kg body weight, about 10 μg/kg body weight, about 50 μg/kg body weight, about 100 μg/kg body weight, about 100 μg/kg body weight per administration. About 200 μg/kg body weight, about 350 μg/kg body weight, about 500 μg/kg body weight, about 1 mg/kg body weight, about 5 mg/kg body weight, about 10 mg/kg body weight, about 50 mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kg body weight, about 350 mg/kg body weight, about 500 mg/kg body weight to about 1000 mg/kg body weight or more and any range derived therefrom. In non-limiting examples of ranges derived from numbers listed herein, about 5 mg/kg body weight to about 100 mg/kg body weight, about 5 μg/kg body weight to about 500 mg/kg body weight can be administered based on the above numbers dose within the range. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 5.0 mg/kg, or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, eg, weekly or every three weeks (eg, such that the patient receives from about 2 to about 20, or eg, about 6 doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimens can be used. The progress of this therapy is easily monitored through conventional techniques and analysis.

The antibodies of the present invention will generally be used in amounts that will achieve the intended purpose. For the treatment or prevention of disease conditions, the antibodies of the present invention or pharmaceutical compositions thereof are administered or used in an effective amount.

For systemic administration , the effective dose can be estimated initially from in vitro assays such as cell culture assays. Doses can then be formulated in animal models to achieve a range of circulating concentrations that include the _IC50 established in cell culture. Such information can be used to more accurately determine useful doses in humans.

Techniques well known in the art can also be used, based on in vivo data (such as animal models) Estimated initial dose.

Doses and intervals can be adjusted individually to provide plasma concentrations of the antibody sufficient to maintain therapeutic effect. Typical patient doses administered via injection are in the range of about 0.1 to 50 mg/kg/day, with a typical range of 0.5 to 1 mg/kg/day. Therapeutically effective plasma concentrations can be achieved via daily administration of various doses. Concentrations in plasma can be measured, for example, via HPLC.

A therapeutically effective dose of the antibodies of the invention will generally provide therapeutic benefit without causing substantial toxicity. Toxicity and therapeutic efficacy of antibodies can be determined in cell cultures or experimental animals via standard pharmaceutical procedures. Cell culture assays and animal studies can be used to determine the _LD50 (dose lethal to 50% of the population) and _ED50 (dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio _LD50 / _ED50 . Antibodies that exhibit large therapeutic indices are preferred. In one aspect, the antibodies according to the invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of doses suitable for use in humans. The dosage lies preferably within a range of circulating concentrations that include the _ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors (eg, the dosage form employed, the route of administration utilized, the condition of the individual, etc.). The precise formulation, route of administration, and dosage can be selected by the individual physician based on the patient's condition (see, eg, Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Chapter 1, page 1, which is incorporated by reference in its entirety). incorporated herein).

The attending physician of a patient treated with an antibody of the invention will instruct how and when to terminate, interrupt, or adjust administration due to toxicity, organ dysfunction, and the like. Conversely, the attending physician will also know how to adjust treatment to higher levels when clinical response is inadequate (excluding toxicity). In the treatment of the target disease, the size of the administered dose will vary depending on the severity of the disease to be treated, the route of administration, and the like. The severity of the disorder can be assessed in part via, for example, standard prognostic assessment methods. In addition, the dosage, and possibly the frequency of administration, will also vary depending on the age, weight and response of the individual patient.

The (multispecific, e.g., bispecific) antibodies of the invention can be administered in combination with one or more other agents in therapy. For example, an antibody of the invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent administered to treat a condition or disease in a subject in need of such treatment. These additional therapeutic agents may comprise any active ingredient suitable for the particular disease being treated, preferably, they are complementary active ingredients that do not adversely affect each other. In certain aspects, the additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, a cell adhesion inhibitor, a cytotoxic agent, an apoptosis activator, or an agent that increases the sensitivity of cells to apoptosis-inducing agents. In a preferred aspect, the additional therapeutic agent is an anticancer agent, such as a microtubule disrupting agent, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, hormone therapy, a kinase inhibitor, a receptor antagonists, tumor cell apoptosis initiators or anti-angiogenic agents.

These other drugs are suitably present in combination in amounts effective for the intended purpose. Effective amounts of these other agents depend on the amount of antibody used, the type of disorder or treatment, and other factors discussed above. Such antibodies are generally used at the same dosage and route of administration as described herein, or about 1% to 99% of the dosage described herein, or at any dosage and via any route determined empirically/clinically as appropriate .

Such combination therapies mentioned above encompass combined administration (in which two or more therapeutic agents are included in the same or separate compositions), as well as separate administration, in which case the (multispecific, e.g. Administration of the bispecific) antibody can occur before, concurrently with, and/or after administration of the additional therapeutic agent and/or adjuvant. The antibodies of the present invention can also be used in combination with radiation therapy. G. Products

In another aspect of the present invention, there is provided an article of manufacture containing materials useful in the treatment, prevention and/or diagnosis of the above-mentioned disorders. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container may contain a composition, by itself or in combination with another composition effective for treating, preventing, and/or diagnosing a condition, and may have a sterile access port (eg, the container may have a stopper that can be pierced through a hypodermic needle) intravenous solution bag or vial). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the condition of choice. In addition, the article of manufacture may comprise (a) a first container containing a composition therein, wherein the composition comprises an antibody of the invention; and (b) a second container containing a composition therein, wherein the composition comprises other cytotoxic or other therapeutics agent. The article of manufacture in this aspect of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively or additionally, the article of manufacture can further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and glucose solution. From a commercial and user perspective, it may further include other materials including other buffers, diluents, filters, needles and syringes. H. Methods and compositions for diagnosis and detection

In certain aspects, any of the antibodies provided herein can be used to detect the presence or absence of its target (eg, CD3, FolR1) in a biological sample. The term "detection" as used herein encompasses quantitative or qualitative detection. In certain aspects, the biological sample comprises cells or tissue, such as prostate tissue.

In one aspect, an antibody according to the invention is provided for use in a method of diagnosis or detection. In another aspect, a method of detecting the presence or absence of CD3 and FolR1 in a biological sample is provided. In certain aspects, the method comprises: contacting a biological sample with an antibody of the invention under conditions that allow binding of the antibody to CD3 and FolR1, and detecting whether a complex is formed between the antibody and CD3 and FolR1. Such methods can be in vitro or in vivo methods. In one aspect, the antibodies of the invention are used to select individuals suitable for treatment with antibodies that bind CD3 and FolRl, eg, where CD3 and FolRl are biomarkers for selecting patients.

Exemplary diseases that can be diagnosed using the antibodies of the invention include cancer, particularly skin cancer or brain cancer.

In certain aspects, an antibody according to the invention is provided, wherein the antibody is labeled. Labels include, but are not limited to, labels or moieties that are directly detected (eg, fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), and moieties that are indirectly detected (eg, via enzymatic reactions or molecular interactions), such as enzymes or ligands. Exemplary labels include, but are not limited to: radioisotopes32P, ^14C , ^125I , ^{3H and131I} ; ^fluorophores such as rare earth chelates or luciferin and derivatives thereof; rose bengal and derivatives thereof; dansyl; umbelliferone; luciferases such as firefly luciferase and bacterial luciferase (US Pat. No. 4,737,456); luciferin; 2,3-dihydrophthalodione; Horseradish peroxidase (HRP); alkaline phosphatase; beta-galactosidase; glucoamylase; lysozyme; carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose 6-phosphate Hydrogenases; heterocyclic oxidases, such as uricase and xanthine oxidase, used in combination with enzymes using hydrogen peroxide dye precursors such as HRP, lactoperoxidase, or microperoxidase; biotin/antibiotic Vegetarian protein; rotation labeling; phage labeling; stable free radicals, etc. III. Sequence amino acid sequence SEQ ID NO CD3 _original HCDR1 TYAMN 1 CD3- _optimized HCDR1 SYAMN 2 CD3 _original /CD3 _optimized HCDR2 RIRSKYNNYATYYADSVKG 3 CD3 _original HCDR3 HGNFGNSYVSWFAY 4 CD3- _optimized HCDR3 HTTFPSSYVSYYGY 5 CD3 _original VH EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS 6 CD3 _optimized VH EVQLLESGGGLVQPGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSS 7 CD3 _original /CD3 _optimized LCDR1 GSSTGAVTTSNYAN 8 CD3 _original / CD3 _optimized LCDR2 GTNKRAP 9 CD3 _raw /CD3 _optimized LCDR3 ALWYSNLWV 10 CD3 _original /CD3 _optimized VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 11 CD3 _raw IgG HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 12 CD3 _optimized IgG HC EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 13 CD3 _original /CD3 _optimized IgG LC QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 14 TYRP1 HCDR1 DYFLH 15 TYRP1 HCDR2 WINPDNGNTVYAQKFQG 16 TYRP1 HCDR3 RDYTYEKAALDY 17 TYRP1 VH QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYFLHWVRQAPGQGLEWMGWINPDNGNTVYAQKFQGRVTMTADTSTSTSTVYMELSSLRSEDTAVYYCTRRDYTYEKAALDYWGQGTLVTVSS 18 TYRP1 LCDR1 RASGNIYNYLA 19 TYRP1 LCDR2 DAKTLAD 20 TYRP1 LCDR3 QHFWSLPFT twenty one TYRP1 VL DIQMTQSPSSLSASVGDRVTITCRASGNIYNYLAWYQQKPGKVPKLLIYDAKTLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHFWSLPFTFGQGTKLEIK twenty two TYRP1 VH-CH1(EE) – CD3 _original /CD3 _optimized VL-CH1 – Fc (Knosle, PGLALA) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYFLHWVRQAPGQGLEWMGWINPDNGNTVYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCTRRDYTYEKAALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP twenty three TYRP1 VH-CH1(EE) – Fc (hole, PGLALA) QVQLVQSGAEVKKPGASVKVSCKASGFNIKDYFLHWVRQAPGQGLEWMGWINPDNGNTVYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCTRRDYTYEKAALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP twenty four TYRP1 VL-CL(RK) DIQMTQSPSSLSASVGDRVTITCRASGNIYNYLAWYQQKPGKVPKLLIYDAKTLADGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHFWSLPFTFGQGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 25 CD3 _original VH-CL EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYVTHQGLSSPVTKSFNR 26 CD3- _optimized VH-CL EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYVTHQGLSSPVTKSFNR 27 Human CD3 ε Stem – Fc (Knob) – Avi QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVSENCVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 28 Human CD3 delta Stem – Fc (hole) – Avi FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCRSEQLYFQGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 29 Cynomolgus monkey CD3 ε Stem – Fc (Knob) – Avi QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVSENCVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 30 Cynomolgus monkey CD3 delta stem – Fc (hole) – Avi FKIPVEELEDRVFVKCNTSVTWVEGTVGTLLTNNTRLDLGKRILDPRGIYRCNGTDIYKDKESAVQVHYRMSQNCVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 31 Human TYRP1 ECD – Fc (Pesle) – Avi QFPRQCATVEALRSGMCCPDLSPVSGPGTDRCGSSSGRGRCEAVTADSRPHSPQYPHDGRDDREVWPLRFFNRTCHCNGNFSGHNCGTCRPGWRGAACDQRVLIVRRNLLDLSKEEKNHFVRALDMAKRTTHPLFVIATRRSEEILGPDGNTPQFENISIYNYFVWTHYYSVKKTFLGVGQESFGEVDFSHEGPAFLTWHRYHLLRLEKDMQEMLQEPSFSLPYWNFATGKNVCDICTDDLMGSRSNFDSTLISPNSVFSQWRVVCDSLEDYDTLGTLCNSTEDGPIRRNPAGNVARPMVQRLPEPQDVAQCLEVGLFDTPPFYSNSTNSFRNTVEGYSDPTGKYDPAVRSLHNLAHLFLNGTGGQTHLSPNDPIFVLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNRQYNMVPFWPPVTNTEMFVTAPDNLGYTYEIQWPSREFSVPEGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 32 Cynomolgus TYRP1 ECD – Fc (Pesle) – Avi QFPRECATVEALRSGMCCPDLSPMSGPGTDRCGSSSGRGRCEAVTADSRPHSPRYPHDGRDDREVWPLRFFNRTCHCNGNFSGHNCGTCRPGWRGAACDQRVLVVRRNLLDLSKEEKNHFVRALDMAKRTTHPLFVIATRRSEEILGPDGNTPQFENISIYNYFVWTHYYSVKKTFLGAGQESFGEVDFSHEGPAFLTWHRYHLLRLEKDMQEMLQEPSFSLPYWNFATGKNVCDICTDDLMGSRSNFDSTLISPNSVFSQWRVVCDSLEDYDTLGTLCNSTESGPIRRNPAGNVARPMVQRLPEPQDVAQCLEVGLFDTPPFYSNSTNSFRNTVEGYSDPTGKYDPAVRSLHNLAHLFLNGTGGQTHLSPNDPIFVLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNRQYNMVPFWPPVTNTEMFVTAPDNLGYTYEVQWPSREFSVPGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 33 Mouse TYRP1 ECD – Fc (Knob) – Avi QFPRECANIEALRRGVCCPDLLPSSGPGTDPCGSSSGRGRCVAVIADSRPHSRHYPHDGKDDREAWPLRFFNRTCQCNDNFSGHNCGTCRPGWRGAACNQKILTVRRNLLDLSPEEKSHFVRALDMAKRTTHPQFVIATRRLEDILGPDGNTPQFENISVYNYFVWTHYYSVKKTFLGTGQESFGDVDFSHEGPAFLTWHRYHLLQLERDMQEMLQEPSFSLPYWNFATGKNVCDVCTDDLMGSRSNFDSTLISPNSVFSQWRVVCESLEEYDTLGTLCNSTEGGPIRRNPAGNVGRPAVQRLPEPQDVTQCLEVRVFDTPPFYSNSTDSFRNTVEGYSAPTGKYDPAVRSLHNLAHLFLNGTGGQTHLSPNDPIFVLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNRQYNMVPFWPPVTNTEMFVTAPDNLGYAYEVQWPGQEFTVSEGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGLNDIFEAQKIEWHE 34 Fc (hole) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSL 35 EGFRvIII ECD – Avi – His LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSVDGGSPTPPTPGGGSGLNDIFEAQKIEWHEARAHHHHHH 36 EGFRvIII P056.021 HCDR1 SYWIA 37 EGFRvIII P056.021 HCDR2 VIHPYDSDTRYSPSFQG 38 EGFRvIII P056.021 HCDR3 VSRSSYAFDY 39 EGFRvIII P056.021 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFDSYWIAWVRQMPGKGLEWMGVIHPYDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVSRSSYAFDYWGQGTLVTVSS 40 EGFRvIII P056.021 LCDR1 KSSQSVLYSSNNKNYLA 41 EGFRvIII P056.021 LCDR2 WASTRES 42 EGFRvIII P056.021 LCDR3 QQVHSGPPPT 43 EGFRvIII P056.021 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQVHSGPPVTFGQGTKVEIK 44 EGFRvIII P056.052 HCDR1 NYWIG 45 EGFRvIII P056.052 HCDR2 TIYPGDSDRRYSPSFQG 46 EGFRvIII P056.052 HCDR3 VSRSSYAFDY 47 EGFRvIII P056.052 VH EVQLVQSGAEVKKPGESLKISCKGSGYTFMNYWIGWVRQMPGKGLEWMGTIYPGDSDRRYSPSFQGQVTLSADKSISTAYLQWSSLKASDTAMYYCARVSRSSYAFDYWGQGTLVTVSS 48 EGFRvIII P056.052 LCDR1 KSSQSVLYSSNNKNYLA 49 EGFRvIII P056.052 LCDR2 WASTRES 50 EGFRvIII P056.052 LCDR3 QQVHSGPPPT 51 EGFRvIII P056.052 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQVHSGPPVTFGQGTKVEIK 52 EGFRvIII P047.019 HCDR1 SIWIH 53 EGFRvIII P047.019 HCDR2 TIYPGDSDTRYSPSFQG 54 EGFRvIII P047.019 HCDR3 TGPGLAFDY 55 EGFRvIII P047.019 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFPSIWIHWVRQMPGKGLEWMGTIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARTGPGLAFDYWGQGTLVTVSS 56 EGFRvIII P047.019 LCDR1 KSSQSVLYSSNNKNYLA 57 EGFRvIII P047.019 LCDR2 WASTRES 58 EGFRvIII P047.019 LCDR3 QQSYSTPIT 59 EGFRvIII P047.019 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSTPITFGQGTKVEIK 60 EGFRvIII P057.012 HCDR1 NYWIA 61 EGFRvIII P057.012 HCDR2 IIYPDDSDTRYSPSFQG 62 EGFRvIII P057.012 HCDR3 ATNIASGGYFDY 63 EGFRvIII P057.012 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFANYWIAWVRQMPGKGLEWMGIIYPDDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARATNIASGGYFDYWGQGTLVTVSS 64 EGFRvIII P057.012 LCDR1 KSSQSVLWNSNNKNYLA 65 EGFRvIII P057.012 LCDR2 WASKRES 66 EGFRvIII P057.012 LCDR3 QQSYSAPIT 67 EGFRvIII P057.012 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLWNSNNKNYLAWYQQKPGQPPKLLIYWASKRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSAPITFGQGTKVEIK 68 EGFRvIII P057.011 HCDR1 RRWIA 69 EGFRvIII P057.011 HCDR2 IIYPGDSDTRYSPSFQG 70 EGFRvIII P057.011 HCDR3 ATNIASGGYFDY 71 EGFRvIII P057.011 VH EVQLVQSGAEVKKPGESLKISCKGSGYNFGRRWIAWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARATNIASGGYFDYWGQGTLVTVSS 72 EGFRvIII P057.011 LCDR1 KSSQSVLWNSNNKNYLA 73 EGFRvIII P057.011 LCDR2 WASKRES 74 EGFRvIII P057.011 LCDR3 QQSYSAPIT 75 EGFRvIII P057.011 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLWNSNNKNYLAWYQQKPGQPPKLLIYWASKRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYSAPITFGQGTKVEIK 76 EGFRvIII P056.027 HCDR1 NNWIA 77 EGFRvIII P056.027 HCDR2 VIYPGDSDKRYSPSFQG 78 EGFRvIII P056.027 HCDR3 VSRSSYAFDY 79 EGFRvIII P056.027 VH EVQLVQSGAEVKKPGESLKISCKGSGYTFGNNWIAWVRQMPGKGLEWMGVIYPGDSDKRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVSRSSYAFDYWGQGTLVTVSS 80 EGFRvIII P056.027 LCDR1 KSSQSVLYSSNNKNYLA 81 EGFRvIII P056.027 LCDR2 WASTRES 82 EGFRvIII P056.027 LCDR3 QQVHSGPPPT 83 EGFRvIII P056.027 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQVHSGPPVTFGQGTKVEIK 84 EGFRvIII P063.056 HCDR1 SYWIA 85 EGFRvIII P063.056 HCDR2 VIHPYDSDTRYSPSFQG 86 EGFRvIII P063.056 HCDR3 VSRSSYAFDY 87 EGFRvIII P063.056 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFDSYWIAWVRQMPGKGLEWMGVIHPYDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVSRSSYAFDYWGQGTLVTVSS 88 EGFRvIII P063.056 LCDR1 KSSQSVLYSSNNKNYLA 89 EGFRvIII P063.056 LCDR2 WASTRES 90 EGFRvIII P063.056 LCDR3 QQQRDGPPVT 91 EGFRvIII P063.056 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQQRDGPPVTFGQGTKVEIK 92 EGFRvIII P064.078 HCDR1 SYWIA 93 EGFRvIII P064.078 HCDR2 VIHPYDSDTRYSPSFQG 94 EGFRvIII P064.078 HCDR3 VSRLSYALDY 95 EGFRvIII P064.078 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFDSYWIAWVRQMPGKGLEWMGVIHPYDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVSRLSYALDYWGQGTLVTVSS 96 EGFRvIII P064.078 LCDR1 KSSQSVLYSSNNKNYLA 97 EGFRvIII P064.078 LCDR2 WASTRES 98 EGFRvIII P064.078 LCDR3 QQVHSGPPPT 99 EGFRvIII P064.078 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQVHSGPPVTFGQGTKVEIK 100 EGFRvIII P065.036 HCDR1 SYWIA 101 EGFRvIII P065.036 HCDR2 VIHPYDSDTRYSPSFQG 102 EGFRvIII P065.036 HCDR3 VSRSSYALDY 103 EGFRvIII P065.036 VH EVQLVQSGAEVKKPGESLKISCKGSGYSFDSYWIAWVRQMPGKGLEWMGVIHPYDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVSRSSYALDYWGQGTLVTVSS 104 EGFRvIII P065.036 LCDR1 KSSQSVLYSSNNKNYLA 105 EGFRvIII P065.036 LCDR2 WASTRES 106 EGFRvIII P065.036 LCDR3 QQVYSGPPVT 107 EGFRvIII P065.036 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQVYSGPPVTFGQGTKVEIK 108 EGFRvIII VH-CH1(EE) – CD3 _original /CD3 _optimized VL-CH1 – Fc (Knosle, PGLALA) EVQLVQSGAEVKKPGESLKISCKGSGYSFDSYWIAWVRQMPGKGLEWMGVIHPYDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVSRSSYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 109 EGFRvIII VH-CH1(EE) – Fc (hole, PGLALA) EVQLVQSGAEVKKPGESLKISCKGSGYSFDSYWIAWVRQMPGKGLEWMGVIHPYDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARVSRSSYAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 110 EGFRvIII VL-CL(RK) DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQQRDGPPVTFGQGTKVEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 111 CD3 _original VH-CL EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYVTHQGLSSPVTKSFNR 26 CD3- _optimized VH-CL EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYVTHQGLSSPVTKSFNR 27 hCD3 QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI 112 Cynomolgus monkey CD3 QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI 113 hTYRP1 QFPRQCATVEALRSGMCCPDLSPVSGPGTDRCGSSSGRGRCEAVTADSRPHSPQYPHDGRDDREVWPLRFFNRTCHCNGNFSGHNCGTCRPGWRGAACDQRVLIVRRNLLDLSKEEKNHFVRALDMAKRTTHPLFVIATRRSEEILGPDGNTPQFENISIYNYFVWTHYYSVKKTFLGVGQESFGEVDFSHEGPAFLTWHRYHLLRLEKDMQEMLQEPSFSLPYWNFATGKNVCDICTDDLMGSRSNFDSTLISPNSVFSQWRVVCDSLEDYDTLGTLCNSTEDGPIRRNPAGNVARPMVQRLPEPQDVAQCLEVGLFDTPPFYSNSTNSFRNTVEGYSDPTGKYDPAVRSLHNLAHLFLNGTGGQTHLSPNDPIFVLLHTFTDAVFDEWLRRYNADISTFPLENAPIGHNRQYNMVPFWPPVTNTEMFVTAPDNLGYTYEIQWPSREFSVPEIIAIAVVGALLLVALIFGTASYLIRARRSMDEANQPLLTDQYQCYAEEYEKLQNPNQSVV 114 human EGFRvIII LEEKKGNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFFSSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA 115 human EGFR LEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEFKKIKVLGSGAFGTVYKGLWIPEGEKVKIPVAIKELREATSPKANKEILDEAYVMASVDNPHVCRLLGICLTSTVQLITQLMPFGCLLDYVREHKDNIGSQYLLNWCVQIAKGMNYLEDRRLVHRDLAARNVLVKTPQHVKITDFGLAKLLGAEEKEYHAEGGKVPIKWMALESILHRIYTHQSDVWSYGVTVWELMTFGSKPYDGIPASEISSILEKGERLPQPPICTIDVYMIMVKCWMIDADSRPKFRELIIEFSKMARDPQRYLVIQGDERMHLPSPTDSNFYRALMDEEDMDDVVDADEYLIPQQGFF SSPSTSRTPLLSSLSATSNNSTVACIDRNGLQSCPIKEDSFLQRYSSDPTGALTEDSIDDTFLPVPEYINQSVPKRPAGSVQNPVYHNQPLNPAPSRDPHYQDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQKGSHQISLDNPDYQQDFFPKEAKPNGIFKGSTAENAEYLRVAPQSSEFIGA 116 hIgG1 Fc region DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGSPFSCSVMHEALHNHY 117 linker GGGGSGGGGS 118 linker DGGGGSGGGGS 119 Human kappa CL domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 120 Human λ CL domain QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 121 Human IgG1 heavy chain constant region (CH1-CH2-CH3) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 122 Bispecific CD3/FolR1 antibody sequence:

According to Kabat's CDR definition FOLR1_16D5IgG_PGLALA VH EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSS 123 CDRH1 NAWMS 124 CDRH2 RIKSKTDGGTTDYAAPVKG 125 CDRH3 PWEWSWYDY 126 VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 11 CDRL1 GSSTGAVTTSNYAN 8 CDRL2 GTNKRAP 9 CDRL3 ALWYSNLWV 10 CD3 _optimized IgG_PGLALA VH EVQLLESGGGLVQPGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSS 7 CDRH1 SYAMN 2 CDRH2 RIRSKYNNYATYYADSVKG 3 CDRH3 HTTFPSSYVSYYGY 5 VL QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVL 11 CDRL1 GSSTGAVTTSNYAN 8 CDRL2 GTNKRAP 9 CDRL3 ALWYSNLWV 10 FOLR1 TCB 16D5-CD3 _optimized 2+1 canonical form K chain EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 127 H chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 128 common light chain QAVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAFRGLIGGTNKRAPGTPARFSGSLLGGKAALTLSGAQPEDEAEYYCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 129 FOLR1 TCB 16D5-CD3 _optimized 2+1 inversion K chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 130 H chain FOLR1 TCB 16D5-CD3 cl22 2+1 Typical form 128 L chain FOLR1 TCB 16D5-CD3 cl22 2+1 Typical form 129 FOLR1 TCB 16D5-CD3 _optimized 1+1 head-to-tail inversion K chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 131 H chain DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ 132 L chain FOLR1 TCB 16D5-CD3 cl22 2+1 Typical form 129 FOLR1 TCB 16D5-CD3 _optimized 1+1 head-to-tail canonical form K chain EVQLLESGGGLVQPGGSLRLSCAASGFQFSSYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCVRHTTFPSSYVSYYGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 133 H chain DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ 134 L chain FOLR1 TCB 16D5-CD3 cl22 2+1 Typical form 129 FOLR1 TCB 16D5-CD3 _optimized 1+1 IgG -like format K chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 135 H chain EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTPWEWSWYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP 136 L chain FOLR1 TCB 16D5-CD3 cl22 2+1 Typical form 129 hu FolR1 MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSLLLLEVANYSRGSGRCIQMWFDAW SLALLLMEVANYSRGSGRCIQMWFDAWSLALLMW 137 IV. Examples

The following are examples of methods and compositions of the present invention. It should be understood that various other aspects may be implemented in light of the general description given above. Example 1 - Production of optimized CD3 binders

Starting from a CD3 binder described previously (see eg WO 2014/131712, which is incorporated herein by reference), referred to herein as "CD3 _primitive " and comprising the VH and VL sequences of SEQ ID NOs: 6 and 11, respectively , we aimed to optimize the properties of this binder by removing two asparagine deamidation sequence motifs at Kabat positions 97 and 100 of the heavy chain CDR3.

For this purpose, we generated an antibody repertoire of heavy chains suitable for phage display, in which the two asparagine at Kabat positions 97 and 100 were removed, and in addition the CDRs H1, H2 and H3 were also randomly assigned to compensate for the loss by affinity The maturation process replaces Asn97 and Asn100 with loss of affinity.

This library was placed on filamentous phage via fusion to the minor coat protein p3 (Marks et al. (1991) J Mol Biol 222 , 581-597) and combined for binding to recombinant CD3ɛ.

Ten candidate clones were identified in a preliminary screen showing acceptable binding to the recombinant antigen as Fab fragments (produced in E. coli) as measured by SPR.

However, after conversion to the IgG format, only one of these clones displayed acceptable binding activity to cells expressing CD3 as measured by flow cytometry.

The selected clones are referred to herein as "CD3 _optimized " and comprise the VH and VL sequences of SEQ ID NOs: 7 and 11, respectively, were further evaluated and converted to bispecific formats as described below. Example 2 - Optimizing the binding of CD3 binders to CD3 and recombinant CD3

For the optimized CD3 binder "CD3 _optimized " and the original CD3 binder "CD3 _original ", both in human IgG1 format with the P329G L234A L235A ("PGLALA", EU numbering) mutation in the Fc region (SEQ ID NO: 12 and 14 (CD3 _original ) and SEQ ID NOs: 13 and 14 (CD3 _optimized )), binding to recombinant CD3 was determined by surface plasmon resonance (SPR).

To assess the effect of deamidation site removal and its effect on antibody stability, native and optimized CD3 binders were tested for binding to recombinant CD3 after 14 days of temperature stress at 37°C or 40°C. Samples stored at -80°C were used as reference. Reference samples and samples stressed at 40°C were in 20 mM His, 140 mM NaCl, pH 6.0, and samples stressed at 37°C were in PBS, pH 7.4, all at 1.2-1.3 mg/ml. After the stress period (14 days), samples in PBS were dialyzed back to 20 mM His, 140 mM NaCl, pH 6.0 for further analysis.

The relative activity concentration (RAC) of the samples was determined by SPR as follows.

SPR was performed on a Biacore T200 instrument (GE Healthcare). Anti-Fab capture antibody (GE Healthcare, #28958325) was immobilized on Series S Sensor Chip CM5 (GE Healthcare) using standard amine coupling chemistry, resulting in a surface density of 4000 – 6000 resonance units (RU). Use HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) as running and dilution buffer. CD3 antibody at a concentration of 2 μg/ml was injected at a flow rate of 5 μl/min for 60 seconds. CD3 antibody (see below) at a concentration of 10 μg/ml was injected for 120 seconds and dissociation was monitored for 120 seconds at a flow rate of 5 μl/min. The wafer surface was regenerated by two consecutive injections of 10 mM glycine pH 2.1 for 60 seconds each. The bulk refractive index difference was corrected by subtracting the blank injection and by subtracting the response obtained from the blank control flow cell. For evaluation, binding reactions were performed 5 s after the end of the injection. To normalize the binding signal, CD3 binding was divided by the anti-Fab reaction (signal (RU) obtained after capture of CD3 antibody on immobilized anti-Fab antibody). Relative activity concentrations were calculated by comparing each temperature stressed sample to the corresponding unstressed sample.

The antigen used was a heterodimer of CD3 delta and CD3 epsilon ectodomains fused to a human Fc domain (see SEQ ID NOs: 28 and 29) with a knob-hole modification and a C-terminal Avi-tag.

The results of this experiment are shown in FIG. 2 . As can be seen, the optimized CD3 binder CD3 _original showed significantly increased binding to CD3 after temperature stress (2 weeks at 37°C, pH 7.4) compared to the original CD3 binder CD3 _optimized . This result confirms that deamidation site removal was successful and correlated with in vivo half-life and formulation of antibodies at neutral pH, resulting in antibodies with excellent stability properties. Binds to CD3 on Jurkat cells

For the optimized CD3 binder "CD3 _optimized " and the original CD3 binder "CD3 _original ", both in human IgG1 format with the P329G L234A L235A ("PGLALA", EU numbering) mutation in the Fc region (SEQ ID NO: 12 and 14 (CD3 _original ) and SEQ ID NOs: 13 and 14 (CD3 _optimized ), binding to CD3 on the human reporter T cell line Jurkat NFAT was determined by FACS.

The Jurkat-NFAT reporter cell (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) is a human acute lymphoblastic leukemia reporter cell line with the NFAT promoter expressing human CD3. Cells were cultured in RPMI1640, 2 g/l glucose, 2 g/l NaHCO ₃ , 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1 × NEAA, 1 × sodium pyruvate at 100,000 to 500,000 cells. Hygromycin B was added at a final concentration of 200 µg/ml whenever cells were passaged.

For binding assays, Jurkat NFAT cells were harvested, washed with PBS and resuspended in FACS buffer. Antibody staining was performed in 96-well round dishes. Therefore, seed 100'000 to 200'000 cells per well. The plate was centrifuged at 400 x g for 4 minutes and the supernatant was removed. Test antibodies were diluted in FACS buffer and 20 μl of antibody solution was added to cells over 30 minutes at 4°C. To remove unbound antibody, cells were plated with diluted secondary antibody (PE Conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human IgG Fcg Fragment Specific; Jackson ImmunoResearch #109-116-170) Wash twice with FACS buffer. After 30 min incubation at 4°C, unbound secondary antibody was washed away. Prior to measurement, cells were resuspended in 200 μl of FACS buffer and then analyzed by flow cytometry using a BD Canto II device.

As shown in Figure 3, the optimized CD3 binder "CD3 _optimized " and the original CD3 binder "CD3 _original " bound CD3 on Jurkat cells equally well. Example 3 - Optimizing the functional activity of CD3 binders

The functional activity of the optimized CD3 binder "CD3 _optimized " was tested in a Jurkat reporter cell assay and compared to the activity of the original CD3 binder "CD3 _original ". To test the functional activity of IgG, anti-PGLALA expressing CHO cells were co-incubated with Jurkat NFAT reporter cells in the presence of increasing concentrations of CD3 _optimized human IgG1 _PGLALA or CD3 native human IgG1 PGLALA. Activation of CD3 on Jurkat NFAT reporter cells following T cell cross-linking induces luciferase production, and luminescence can be measured as an activation marker. CD3 native human IgG1 wt was included as a negative control, which failed to bind to anti- _PGLALA expressing CHO cells and thus failed to cross-link on Jurkat NFAT cells. A schematic diagram of the analysis is provided in FIG. 4 .

Anti-PGLALA expressing CHO cells are CHO-K1 cells engineered to express antibodies on their surface that specifically bind human IgGi Fc ( _PGLALA ) (see WO 2017/072210, which is incorporated herein by reference). These cells were cultured in DMEM/F12 medium containing 5% FCS + 1% GluMax. Jurkat NFAT reporter cells are described in Example 2.

Upon simultaneous binding of CD3 huIgG1 PGLALA with CHO expressed on Jurkat-NFAT reporter cells and anti-PGLALA expressed on CD3, the NFAT promoter was activated and resulted in the expression of active firefly luciferase. The intensity of the luminescent signal (obtained by the addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Jurkat-NFAT reporter cells were grown in suspension and incubated in RPMI1640, 2 g/l glucose, 2 g/l NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1 × NEAA, 1 × acetone 100,000 to 500,000 cells per ml, 200 µg hygromycin per ml. For analysis, CHO cells were harvested and viability was determined using ViCell. Plate 30 000 target cells/well in flat-bottomed white-walled 96-well plates (Greiner bio-one #655098) and 100 µl of medium, and 50 µl/well of diluted antibody or medium (for control) added to CHO cells. Subsequently, Jurkat-NFAT reporter cells were collected and viability was assessed using ViCell. Cells were resuspended at 1.2 million cells/ml in cell culture medium without hygromycin B and added to CHO cells at 60,000 cells/well (50 μl/well) to obtain a 2:1 final effector Ratio to target (E:T) and final volume of 200 μl/well. Subsequently, 4 µl of GloSensor (Promega #E1291) was added to each well (2% of final volume). Incubate cells at 37°C for 24 hours in a humidified incubator. At the end of the incubation period, use the TECAN Spark 10M to detect luminescence.

As shown in Figure 5, the optimized CD3 binder CD3 _original had similar activity on Jurkat NFAT cells after _optimized cross-linking as CD3. Example 4 - Generation of T Cell Bispecific Antibodies Comprising Optimized CD3 Binders TYRP1 TCB

The optimized CD3 binders identified in Example 1 ("CD3 _optimized ", SEQ ID NOs: 7 (VH) and 11 (VL)) were used to generate T cell bispecific antibodies (TCBs) targeting CD3 and TYRP1 ("CD3 optimized") TYRP1 TCB”).

The TYRP1 binder included in this TCB was generated by humanizing the TYRP1 binder "TA99" (see GenBank entries AXQ57811 and AXQ57813 for heavy and light chains, respectively), and comprises SEQ ID NO: 18 and Heavy and light chain variable region sequences shown in 22.

A schematic diagram of the TCB molecule is provided in Figure 6 and its full sequence is given as SEQ ID NOs: 23, 24, 25 and 27.

Similar molecules were also prepared with the original CD3 binding sequences (SEQ ID NOs: 23, 24, 25 and 26).

Bispecific molecules were produced by transient transfection of HEK293 EBNA cells. Cells were transfected with the corresponding expression vector at a ratio of 1:2:1:1 ("Vector heavy chain (VH-CH1-VL-CH1-CH2-CH3)": "Vector light chain (VL-CL)": "Vector heavy chain (VH-CH1-CH2-CH3)": "Vector light chain (VH-CL)"). The cells were centrifuged and the medium was replaced with pre-warmed CD CHO medium (Thermo Fisher, #10743029). The expression vector was mixed in CD CHO medium, polyethylenimine (PEI, Polysciences, #23966-1) was added, the solution was vortexed and incubated at room temperature for 10 minutes. Cells (2 million/ml) were then mixed with the carrier/PEI solution, transferred to flasks, and incubated for 3 hours at 37°C in a shaking incubator under a 5% CO ₂ atmosphere. After incubation, Excell medium with supplements (80% of the total volume) was added. One day after transfection, supplements (feed, 12% of total volume) were added. Cell supernatants were collected after 7 days by centrifugation and subsequent filtration (0.2 μm filter).

Proteins were purified from filtered cell culture supernatants using standard methods. Briefly, Fc-containing proteins were purified from cell culture supernatants via protein A-affinity chromatography (MabSelect Sure, GE Healthcare: equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; Elution buffer: 20 mM sodium citrate, 100 mM NaCl, 100 mM glycine pH 3.0). Elution was done at pH 3.0, and the pH of the sample was immediately neutralized. Proteins were concentrated by centrifugation (Millipore Amicon® ULTRA-15 (#UFC903096)) and analyzed by particle size chromatography (Superdex 200, GE Healthcare) in 20 mM histidine, 140 mM sodium chloride, pH 6.0 The aggregated proteins are separated from the monomeric proteins.

The concentration of purified protein was determined by measuring the absorbance at 280 nm using the mass extinction coefficient calculated according to Pace et al. (1995), Protein Science 4, 2411-23 based on the amino acid sequence. Protein purity and molecular weight were analyzed by CE-SDS in the presence and absence of reducing agent using LabChipGXII (Perkin Elmer) (Table 1). Use either equilibrated in running buffer ( ₂₅ mM _K2HPO4 , 125 mM NaCl, 200 mM L-arginine monohydrochloride, pH 6.7 or 200 mM _KH2PO4 , 250 mM KCl pH 6.2, respectively ₎ Analytical size exclusion columns (TSKgel G3000 SW XL or UP-SW3000) were used for the determination of aggregate content by HPLC chromatography at 25°C (Table 2). Table 1. CE-SDS analysis of TYRP1 TCB (non-reduced). molecular Crest number Size [kDa] purity[%] TYRP1 TCB CD3 _optimization 1 221 100 TYRP1 TCB CD3 _original 1 206 100 Table 2. Summary production and purification of TYRP1 TCBs. molecular Titer [mg/l] Recovery rate[%] Yield [mg/L] Analytical SEC (HMW/monomer/LMW) [%] TYRP1 TCB CD3 _optimization 114 20 22.8 0.5/ 98.6 /0.9 TYRP1 TCB CD3 _original 72 12 8.7 0/ 97.5 /2.5 Example 5 - Binding of T cell bispecific antibodies comprising optimized CD3 binders to CD3 and TYRP1 and to recombinant CD3

Binding of TYRP1 TCB to recombinant CD3 was assessed by SPR using TCB with optimized (TYRP1 TCB CD3 _optimized ) or original (TYRP1 TCB CD3 _original ) CD3 binding sequences.

SPR experiments were performed on a Biacore T200 with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% (v/v) Surfactant P20 (GE Healthcare)).

TYRP1 TCBs were captured on the surface of a CM5 sensor wafer with immobilized antibodies that specifically bind human IgGi Fc ( _PGLALA ) (see WO 2017/072210, which is incorporated herein by reference). The capture antibody was coupled to the sensor wafer surface by direct immobilization at pH 5.0 at approximately 8700 resonance units (RU) using a standard amine coupling kit (GE Healthcare). TCB molecules were captured at 5 nM for 30 seconds at a flow rate of 10 μl/min.

Human and cynomolgus monkey antigens (see below) were passed through flow-through cells at concentrations of 12.35 – 3000 nM at a flow rate of 30 μl/min for 240 seconds. The dissociation phase was monitored for 240 seconds and triggered by changing the sample solution to HBS-EP. After each cycle, use one injection of 10 mM glycine pH 2.0 for 30 s to regenerate the wafer surface.

The antigens used were heterodimers of human or cynomolgus monkey CD3 delta and CD3 epsilon ectodomains with a human Fc domain with a knob-hole modification and a C-terminal Avi-tag (see SEQ ID NOs: 28 and 29). (human CD3) and SEQ ID NOs: 30 and 31 (cynomolgus monkey CD3)) fusion.

Bulk refractive index differences were corrected by subtracting the response obtained on the reference flow cell (not captured by the TCB). Affinity constants were derived from kinetic rate constants by fitting 1:1 Langmuir binding using BIAeval software (GE Healthcare).

The _KD values for binding to human and cynomolgus CD3 were determined to be 50 nM and 20 nM _optimized for TYRP1 TCB CD3, respectively, and were similar to the _original values of TYRP1 TCB CD3 (50 nM and 40 nM, respectively).

This shows that the two TCBs comprising CD3- _optimized or CD3- _naive bind equally well to recombinant CD3 under unstressed conditions.

Binding of TYRP1 TCBs to recombinant human CD3 was also assessed after 14 days of temperature stress at 37°C or 40°C using TCBs with optimized or original CD3 binding sequences. The experiments were performed as described in Example 2 above, using TCB instead of IgG molecules.

The results of this experiment are shown in Figure 7.

As can be seen in Figure 7, the TCB containing the optimized CD3 binder CD3 _optimized showed significant binding to CD3 after stress (2 weeks at 37°C, pH 7.4) compared to the TCB containing the original CD3 binder CD3 _original Increase. This result confirms that the improved properties of the optimized CD3 binder (see Example 2) are maintained at TCB levels. Binding to recombinant TYRP1

Binding to recombinant TYRP1 was assessed by SPR using TYRP1 Fab fragments prepared by cytoplasmin digestion of the corresponding antibodies.

SPR experiments were performed on a Biacore T200 with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% (v/v) Surfactant P20 (GE Healthcare)).

Antibodies that specifically bind human IgGi Fc ( _PGLALA ) (see WO 2017/072210, which is incorporated herein by reference) were coupled directly to the CM5 sensor at pH 5.0 using a standard amine coupling kit (GE Healthcare) on the wafer. Antibody was captured at a flow rate of 10 µl/min for 30 seconds (see below). A 3-fold dilution series of the TYRP1 Fab fragment was passed over a flow cell at 30 µl/min for 180 seconds and the associated phase was recorded. The dissociation phase was monitored for 180 seconds or 1200 seconds and triggered by changing the sample solution to HBS-EP. After each cycle, the wafer surface was regenerated with an injection of 10 mM glycine pH 2 at 30 μl/min for 30 seconds.

The antigens used were human, cynomolgus monkey or mouse TYRP1 extracellular domain (ECD) and human Fc-domain with knob-hole (and PG LALA) modifications and a C-terminal Avi-tag (see SEQ ID NOs: 32 and 35 (human). TYRP1), SEQ ID NOs: 33 and 35 (cynomolgus TYRP1) or monomeric fusions of SEQ ID NOs: 34 and 35 (mouse TYRP1)).

The bulk refractive index difference was corrected by subtracting the response obtained on the reference flow cell (where no antigen was captured). Affinity constants (K _D ) were derived from kinetic rate constants by fitting 1:1 Langmuir binding using BIAeval software (GE Healthcare).

_KD values for binding to human, cynomolgus monkey, and mouse TYRP1 were determined to be 130 pM, 180 pM and 530 pM, respectively, and were similar to those of the parental TA99 antibody (90 pM, 120 pM, and 310 pM, respectively) .

Binding of TYRP1 TCBs to TYRP1 was also assessed after 14 days of temperature stress at 37°C or 40°C using TCBs with optimized or original CD3 binding sequences.

Experiments were performed as above for binding to CD3 using recombinant TYRP1 (Sino Biologicals) as antigen.

The results of this experiment are shown in FIG. 8 . These results demonstrate that binding to human TYRP1 (and the corresponding TYRP1 binder in the IgG format) of both TCBs is not affected by stress conditions. Binds to CD3 on Jurkat cells

As described above in Example 2, binding of CD3 on the human reporter T cell line Jurkat NFAT was determined by FACS of TYRP1 TCB comprising the optimized CD3 binder "CD3 _optimized " or the original CD3 binder "CD3 _original ".

As shown in Figure 9, TCB containing the optimized CD3 binder "CD3 _optimized " bound at least as well to CD3 on Jurkat cells as TCB containing the original CD3 binder "CD3 _original ". Example 6 - Functionally active CD3 activation of T cell bispecific antibodies comprising optimized CD3 binders

TYRP1 TCB containing optimized CD3 binder CD3 _optimized or original CD3 binder CD3 _original (Example 4) in TYRP1 positive melanoma cells M150543 (primary melanoma cell line) in Jurkat NFAT reporter cell assay (see Example 3) , obtained from the Dermatology Cell Bank of the University of Zurich) was tested in the presence of .

Upon simultaneous binding of TYRP1 TCB to TYRP1-positive target cells and CD3 antigen (expressed on Jurkat-NFAT reporter cells), the NFAT promoter is activated and results in the expression of active firefly luciferase. The intensity of the luminescent signal (obtained by the addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signaling. Analysis was performed as described in Example 3 using M150543 instead of anti-PGLALA expressing CHO cells.

As seen with IgG (Example 3), both TCBs containing CD3- _optimized or CD3- _naive had similar functional activity on Jurkat NFAT reporter cells and included CD3 activation in a concentration-dependent manner (Figure 10). target cell killing

In the next step, both TCB molecules were tested in tumor cell toxicity assays using freshly isolated human PBMCs from three different donors co-incubated with the human melanoma cell line M150543. T cell lysis was measured by LDH release after 24 hours and 48 hours. Activation of CD4 and CD8 T cells was analyzed after 48 hours by upregulating CD69 and CD25 on both cell subsets.

Briefly, target cells were harvested with trypsin/EDTA, washed, and plated at a density of 30,000 cells/well using flat-bottomed 96-well dishes. Cells were allowed to adhere overnight. Peripheral blood mononuclear cells (PBMC) were prepared by Histopaque density centrifugation of fresh blood obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered in a Histopaque gradient (Sigma, #H8889). After centrifugation (450 xg, 30 min, room temperature), the plasma above the mesophase containing PBMCs was discarded, and the PBMCs were transferred to new Falcon tubes, which were then filled with 50 ml PBS. The mixture was centrifuged (400 xg, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was washed twice with sterile PBS (centrifugation step 350 xg, 10 min). The resulting PBMC populations were automatically counted (ViCell) and stored in a cell incubator at 37°C, 5% CO ₂ in RPMI1640 medium containing 10% FCS and 1% FCS until further use (no more than 24 hours) L-propylamine-L-glutamine (Biochrom, #K0302). For poisoning assays, antibodies were added at the indicated concentrations (triplicates). PBMCs were added to target cells to obtain a final effector to target (E:T) ratio of 10:1. Assessed by quantifying LDH released from apoptotic/necrotic cells into cell supernatant (LDH detection kit, Roche Applied Science, #11 644 793 001) after 24 hours of incubation at 37°C, 5% CO ₂ Target cell poisoning. Maximal lysis of target cells (= 100%) was achieved by incubating target cells with 1% Triton X-100. Minimal lysis (= 0%) refers to target cells co-incubated with effector cells without the bispecific construct.

CD8 and CD4 T cell activation following TCB-mediated T cell killing of target cells was assessed by flow cytometry using antibodies that recognize the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). After 48 hours of incubation, PBMCs were transferred to round bottom 96-well plates, centrifuged at 350 x g for 5 minutes, and washed twice with FACS buffer. Surface staining for CD4 APC (BioLegend #300514), CD8 FITC (BioLegend #344704), CD25 BV421 (BioLegend #302630) and CD69 PE (BioLegend #310906) was performed according to the supplier's instructions. Cells were washed twice with 150 μl/well FACS buffer and fixed with 100 μl/well Fixation Buffer (BD #554655) for 15 minutes at 4°C. After centrifugation, samples were resuspended in 200 μl/well FACS buffer. Samples were analyzed with BD FACS Fortessa.

On all three donors, both TCBs containing optimized or original CD3 binders induced T cell activation and, in a comparable manner, tumor cell lysis (Figure 11). The EC50 values for tumor cell lysis after 48 hours for all three donors are summarized in Table 3. Table 3. Summary of EC50 values for tumor cell killing with TYRP1 TCB at 48 hours. PBMC donor TYRP1 TCB (CD3 _optimized ) EC50 (95% confidence interval ) TYRP1 TCB ( CD3 _raw ) EC50 (95% confidence interval ) Donor 1 0.31 nM (0.17 to 0.55) 0.44 nM (0.28 to 0.70) Donor 2 0.03 nM (0.02 to 0.06) 0.05 nM (0.02 to 0.09) Donor 3 0.08 nM (0.07 to 0.1) 0.14 nM (0.11 to 0.18) Example 7 - PK studies of T cell bispecific antibodies in mice

Intravenous bolus injection at 1 mg/kg to human FcRn transgenic mice (line 32, homozygous) and FcRn knockout mice (Jackson Laboratory line numbers 003982 and 014565) (n=3/strain/test compound) ) The pharmacokinetics (PK) of TYRP1 TCB with different CD3 binders (CD3 _native and CD3 _optimized ) were then investigated. Serial blood microsamples from human FcRn transgenic (tg) mice up to 672 hours (9 samples per mouse from 5 minutes to 672 hours post-dose) and up to 96 hours in FcRn knockout (ko) mice (dosed 8 samples per mouse) were obtained from 5 min to 96 h post-dose. Serum was prepared and stored frozen until analysis. Mouse serum samples were analyzed under non-GLP conditions using the cobas® e411 (Roche) instrument using the genetic ECLIA method specific for human Ig/Fab CH1/κ domains. Pharmacokinetic evaluations were performed using standard non-compartmental analysis.

The results are shown in Table 4. This shows that engineering of the CDRs does not give rise to additional sequence responsibilities that would affect antibody clearance. In terms of serum half-life, CD3 _optimization was as good as CD3 _original , with the additional benefit of increased CDR stability. Table 4. Clearance data for huFcRn tg mice and FcRn ko mice (ml/day/kg; mean and (CV)). mouse strain TYRP1 TCB (CD3 _original ) TYRP1 TCB (CD3 _optimized ) hFcRn tg32 8.82 (10.0) 6.68 (12.5) FcRn ko 66.4 (16.2) 65.0 (6.5) Example 8 - Generation of another T cell bispecific antibody comprising an optimized CD3 binder

The optimized CD3 binders identified in Example 1 ("CD3 _optimized ", SEQ ID NOs: 7 (VH) and 11 (VL)) were used to generate T cell bispecific antibodies (TCBs) targeting CD3 and EGFRvIII ("CD3 optimized") EGFRvIII TCB”).

The EGFRvIII binder contained in this TCB (P063.056) was derived from phage display followed by affinity maturation (see below) and comprises the heavy and light chain variable region sequences shown in SEQ ID NOs: 88 and 92, respectively .

A schematic diagram of the TCB molecule is provided in Figure 6 and its full sequence is given as SEQ ID NOs 109, 110, 111 and 27.

Similar molecules were also prepared with the original CD3 binding sequences (SEQ ID NOs 109, 110, 111 and 26).

Bispecific molecules were produced by transient transfection of HEK293 EBNA cells, purified and analyzed as described above in Example 4.

In addition, EGFRvIII antibodies derived from phage display were produced in a similar fashion (HEK EBNA cells transfected with expression vectors for IgG heavy and light chains in a ₁ :1 ratio) in human IgGl format and used as described below.

All IgG and TCB constructs were purified to comparable quality with a monomer content greater than 95% as determined by particle size sieve chromatography. EGFRvIII antibody selection

The EGFRvIII antibody was derived from phage display and affinity matured. Displays high affinity binding and binding to EGFRvIII (P056.021 (SEQ ID NO: 40 and 44), P056.052 (SEQ ID NO: 48 and 52), P047.019 (SEQ ID NO: 56 and 60), P057.012 (SEQ ID NO: 64 and 68), P057.011 (SEQ ID NO: 72 and 76), P056.027 (SEQ ID NO: 80 and 84)) specific antibodies using CHO cells stably expressing EGFRvIII and EGFRvIII The positive human glioblastoma cell line DK-MG was tested for binding to EGFRvIII expressed on the cell surface. To confirm specificity, and to exclude cross-reactivity with wild-type EGFR (EGFRwt), selected antibodies were tested for binding to the EGFRwt-positive human tumor cell line MKN-45 (Figure 12). Cetuximab was included as a positive control for binding to EGFRwt and non-targeting DP47 IgG as a negative control. All selected antibodies specifically bound EGFRvIII without cross-reactivity with EGFRwt and were considered for further characterization.

In the next step, the functional activity of these EGFRvIII antibodies as IgG1 PGLALA (human IgG1 form with P329G L234A L235A (“PGLALA”, EU numbering) mutation in the Fc region) was assessed on DK-MG cells by measuring luminescence , these DK-MG cells were co-incubated with Jurkat NFAT reporter cells expressing the anti-PGLALA chimeric antibody receptor (CAR) (CAR J assay, see PCT Application No. PCT/EP2018/086038, which is incorporated by reference in its entirety This article). DP47 IgG1 PGLALA was included as a negative control. All tested EGFRvIII antibodies induced strong activation of CAR-expressing Jurkat NFAT reporter cells (Figure 13). All tested EGFRvIII antibodies were selected for conversion to TCB format (with CD _orig as CD3 binder) except for P047.019 which showed the weakest binding and activation.

The binding of selected EGFRvIII antibodies converted to TCB format to CHO-EGFRvIII cells was compared with that of the corresponding IgG (Figure 14), confirming that conversion to TCB format had no effect on the binding capacity of EGFRvIII antibodies. Most of the tested EGFRvIII clones retained their ability to bind to EGFRvIII after conversion to TCB format; only clone P057.011 displayed slightly reduced binding to EGFRvIII in TCB format compared to the corresponding IgG (Table 5). Table 5. Binding of EGFRvIII IgG and TCB to CHO-EGFRvIII (EC50). EGFRvIII pure line EC50IgG (nM) EC50 TCB (nM) P056.021 16.5 13.0 P056.027 13.1 15.9 P056.052 18.2 19.5 P057.012 3.0 5.5 P057.011 5.3 12.8

Subsequently, the functional activity of EGFRvIII TCB was tested in a Jurkat NFAT reporter cell assay on EGFRvIII positive DK-MG cells (Figure 15). All tested EGFRvIII TCBs were active in the Jurkat NFAT reporter cell assay, with P056.021 being the most potent, followed by P056.027, P056.052 and P057.012 with similar activity, and P057.011 with the least activity. Next, EGFRvIII TCBs were tested in tumor cell lysis assays with PBMCs co-cultured with DK-MG or MKN-45 cells to rule out cross-reactivity of EGFRvIII TCBs with EGFRwt (Figure 16). In this assay, in addition to tumor cell lysis, T cell activation (Figure 17) and cytokine release (Figure 18) were also measured as additional readouts. As seen in the previous reporter cell analysis, EGFRvIII TCB P056.021 had the highest activity against EGFRvIII positive cells, but did not have any activity against EGFRwt positive cells. EGFRvIII TCB P057.011 showed no specific activity on EGFRwt cells and was therefore excluded. EGFRvIII TCBs P056.027, P056.052 and P057.012 had comparable activities. Based on these results, EGFRvIII binders P056.021 and P057.012 were selected for another round of affinity maturation.

No good binder could be derived from P057.012 (results not shown). The affinity and specificity of selected EGFRvIII binders derived from P056.021 for EGFRvIII determined by SPR are shown in Table 6. Table 6. Affinity and specificity of selected EGFRvIII binders for EGFRvIII as determined by SPR. binder Specificity ( no binding to EGFRwt ) Binds to EGFRvIII (KD [nM] ) P056.021 (parental) Yes 35 P063.056 Yes 10 P064.078 no 15 P065.036 no 10

Affinity matured EGFRvIII binders (P063.056 (SEQ ID NO: 88 and 92), P064.078 (SEQ ID NO: 96 and 100), P065.036 (SEQ ID NO: 104 and 108)) were also directed against U87MG - Specific binding to EGFRvIII on EGFRvIII and MKN-45 cells was compared to the parental binder (Figure 19). The best EGFRvIII binder in terms of affinity and specificity for EGFRvIII, P063.056, was selected for conversion to TCB format with CD3 _native or CD3 _optimized as CD3 binder.

The functional activity of EGFRvIII TCB P063.056 (CD3 _optimized or CD3 _native ) was compared to the parental EGFRvIII TCB P056.021 in the Jurkat NFAT reporter cell assay on U87MG-EGFRvIII, DK-MG and MKN-45 cells (Figure 20) . All three TCBs induced specific Jurkat NFAT activation only in the presence of EGFRvIII positive cells. EGFRvIII TCB P063.056 had slightly higher activity than the parental EGFRvIII TCB P056.021. method surface plasmon resonance

The affinity of EGFRvIII antibodies for EGFRvIII was determined by surface plasmon resonance on Biacore T200 at 25°C using HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005 % (v/v) surfactant P20; GE Healthcare) assay. Anti-EGFRvIII PGLALA IgG was captured at 25 nM for 30 sec with an antibody immobilized on a CM5 chip that specifically binds to human IgGi Fc ( _PGLALA ) (see WO 2017/072210, which is incorporated herein by reference). EGFRvIII-ECD avi his antigen (see below, Example 9) was passed through all flow cells at a concentration of 12.4 - 1000 nM at a flow rate of 30 μl/min for 200 seconds. The dissociation phase was monitored for 300 seconds and triggered by changing the sample solution to HBS-EP. After each cycle, the wafer surface was regenerated with an injection of 10 mM glycine pH 2.0 for 30 seconds. The bulk refractive index difference was corrected by subtracting the response obtained on the reference flow cell. Affinity constants were derived from kinetic rate constants by fitting 1:1 Langmuir binding using BIAeval software (GE Healthcare).

For specificity, EGFRvIII and EGFRwt ECD antigens were captured with anti-his (Penta His, Qiagen) immobilized on CM5 chips at 100 nM for 40 sec. A single injection of 500 nM of anti-EGFRvIII antibody was performed for 60 seconds, followed by regeneration with 10 mM glycine pH 2.0 for 60 seconds. Response units above 50 were observed for EGFRvIII binding. Responses above 5 Response Units (RU) were considered positive for EGFRwt binding, and IgG was classified as specific for EGFRwt responses below 5 RU. cell line

The Jurkat-NFAT reporter cell (GloResponse Jurkat NFAT-RE-luc2P; Promega #CS176501) is a human acute lymphoblastic leukemia reporter cell line with the NFAT promoter expressing human CD3. Cells were cultured in RPMI1640, 2 g/l glucose, 2 g/l NaHCO ₃ , 10% FCS, 25 mM HEPES, 1 % GlutaMAX, 1 × NEAA, 1 × sodium pyruvate, 100,000-500,000 cells per ml . Hygromycin B was added at a final concentration of 200 µg/ml whenever cells were passaged.

Jurkat NFAT cells with PGLALA CAR were generated in-house. The original cell line (Jurkat NFAT; Signosis) is a human acute lymphoblastic leukemia reporter cell line with the NFAT promoter, resulting in luciferase expression upon activation by human CD3. It is engineered to express a chimeric antigen receptor capable of recognizing the P293G LALA mutation. In culture, cells were grown in suspension in RPMI1640 supplemented with 10% FCS and 1% glutamine and maintained at between 400,000-1.5 million cells per mL.

CHO-EGFRvIII cells are regenerated internally. CHO-K1 cells were stably transduced with EGFRvIII. Cells were grown in DMEM/F12 medium containing 5% FCS, 1% GlutaMAX, and 6 µg/ml puromycin.

DK-MG (DSMZ #ACC 277) is a human glioblastoma cell line. DK-MG cells were enriched by cell sorting for EGFRvIII expression. Cells were cultured in RPMI 1860, 10% FCS, and 1% GlutaMAX.

U87MG-EGFRvIII (ATCC HTB-14) is a human glioblastoma cell line stably transduced with EGFRvIII. Cells were cultured in DMEM, 10% FCS, and 1% GlutaMAX.

MKN-45 (DSMZ ACC 409) is a human gastric adenocarcinoma cell expressing high levels of EGFRwt. Cells were cultured in Advanced RPMI1640 containing 2% FCS and 1% GlutaMAX. Target binding by flow cytometry

Cells for binding experiments were harvested, washed with PBS, and resuspended in FACS buffer. Antibody staining was performed in 96-well round dishes. Cells were collected, counted, and seeded at 100 000 to 200 000 cells per well. The plate was centrifuged at 400 xg for 4 minutes, and the supernatant was removed. Test antibodies were diluted in FACS buffer and 20 μl of antibody solution was added to cells over 30 minutes at 4°C. To remove unbound antibody, add diluted secondary antibody PE-conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human IgG Fcg Fragment specific (Jackson ImmunoResearch, #109-116-170 or #109-116- 098), cells were washed twice with FACS buffer. After 30 min incubation at 4°C, unbound secondary antibody was washed away. Prior to measurement, cells were resuspended in 200 μl of FACS buffer and analyzed by flow cytometry using BD Canto II or BD FACS Fortessa. CAR J NFAT reporter cell analysis using EGFRvIII PGLALA IgG

The potency of EGFRvIII PGLALA IgG to induce T cell activation was assessed using the CAR J NFAT reporter cell assay. The principle of this assay is the co-culture of Jurkat-NFAT engineered effector cells with cancer cells expressing tumor antigens. Only when IgG was mutated through PGLALA and the target antigen EGFRvIII was simultaneously bound to the CAR, the NFAT promoter was activated and resulted in increased luciferase expression in Jurkat effector cells. Upon addition of sufficient substrate, active firefly luciferase induces luminescence, which can be measured as a signal for CAR-mediated activation. Briefly, target cells were harvested and viability was determined. On the day prior to the start of the assay, 30 000 target cells/well were plated in 100 µl of medium in flat-bottomed white wall 96-well dishes (Greiner bio-one, #655098). The next day, the medium was removed and 25 μl/well of diluted antibody or medium (for the control group) was added to the target cells. Subsequently, Jurkat-NFAT reporter cells were collected and viability was assessed using ViCell. Cells were resuspended in cell culture medium without hygromycin B at 1.5 million cells/ml and added to tumor cells at 75,000 cells/well (50 μl/well) to obtain a 2.5:1 final effector Ratio to target (E:T) and final volume of 75 μl/well. 4 μl of GloSensor (Promega, #E1291) was then added to each well (2% of final volume). The cells were incubated at 37°C for 24 hours in a humidified incubator. At the end of the incubation time, the pans were brought to room temperature (approximately 15 minutes). 25 μl/well One-Glo Luciferase (Promega, #E6120) was then added and the plate was incubated in the dark for 15 minutes before luminescence was detected using the TECAN Spark. Jurkat NFAT reporter cell assay with EGFRvIII TCB

The ability of EGFRvIII TCBs with modified CD3 or original CD3 binders to induce T cell cross-linking and subsequent T cell activation was assessed using EGFRvIII positive cells and Jurkat-NFAT reporter cells. Following simultaneous binding of EGFRvIII TCB to EGFRvIII positive target cells and CD3 antigen (expressed on Jurkat-NFAT reporter cells), the NFAT promoter is activated and results in the expression of active firefly luciferase. The intensity of the luminescent signal (obtained by addition of the luciferase substrate) is proportional to the intensity of CD3 activation and signaling. For analysis, target cells were collected and viability was determined. Plate 30 000 cells/well in 96-well flat-bottomed white-walled plates (Greiner Bio-One, no. 655098) in 100 µl of medium, and add 50 µl/well of diluted antibody or medium (for control) to in target cells. Subsequently, Jurkat-NFAT reporter cells were collected and viability was assessed using ViCell. Cells were resuspended in cell culture medium without hygromycin B at 1.2 million cells/ml and added to tumor cells at 60,000 cells/well (50 μl/well) to obtain a 2:1 final effector Ratio to target (E:T) and final volume of 200 μl/well. 4 μl of GloSensor (Promega, #E1291) was then added to each well (2% of final volume). The cells were incubated at 37°C for 24 hours in a humidified incubator. At the end of the incubation period, use the TECAN Spark to detect luminescence. T cell-mediated tumor cell killing

Target cells were harvested with trypsin/EDTA, washed, and plated at a density of 30,000 cells/well using flat-bottomed 96-well dishes. Cells were allowed to adhere overnight. Peripheral blood mononuclear cells (PBMC) were prepared by Histopaque density centrifugation of fresh blood obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered in a Histopaque gradient (Sigma, #H8889). After centrifugation (450 xg, 30 min, room temperature), the plasma above the PBMC-containing mesophase was discarded and the PBMCs were transferred to new falcon tubes, which were subsequently filled with 50 ml PBS. The mixture was centrifuged (400 xg, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was washed twice with sterile PBS (centrifugation step 350 xg, 10 min). The resulting PBMC populations were automatically counted (ViCell) and stored in a cell incubator at 37°C, 5% CO ₂ in RPMI1640 medium containing 10% FCS and 1% FCS until further use (no more than 24 hours) GlutaMAX. For poisoning assays, antibodies (triplicates) were added at the indicated concentrations. PBMCs were added to target cells to obtain a final effector to target (E:T) ratio of 10:1. Assessed by quantification of LDH released from apoptotic/necrotic cells into the supernatant of cells (LDH Detection Kit, Roche Applied Science, #11 644 793 001) after 24 hours of incubation at 37°C, 5% CO ₂ target cell killing. Maximal lysis of target cells (= 100%) was achieved by incubating target cells with 1% Triton X-100. Minimal lysis (= 0%) refers to target cells co-incubated with effector cells without the bispecific construct.

CD8 and CD4 T cell activation following TCB-mediated T cell killing of target cells was assessed by flow cytometry using antibodies that recognize the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). After 48 hours of incubation, PBMCs were transferred to round bottom 96-well plates, centrifuged at 350 x g for 5 minutes, and washed twice with FACS buffer. Surface staining for CD4 APC (BioLegend #300514), CD8 FITC (BioLegend #344704), CD25 BV421 (BioLegend #302630) and CD69 PE (BioLegend #310906) was performed according to the supplier's instructions. Cells were washed twice with 150 μl/well FACS buffer and fixed with 100 μl/well Fixation Buffer (BD, #554655) for 15 minutes at 4°C. After centrifugation, samples were resuspended in 200 μl/well FACS buffer. Samples were analyzed with BD FACS Fortessa.

Cytokinetic secretion in supernatants was measured by flow cytometry using a Cytometry Bead Array (CBA) according to the manufacturer's instructions, but only 25 μl supernatant and beads were used instead of 50 μl beads and sample. The following CBA sets (BD Biosciences) were used: CBA Human Interferon Gamma (IFNγ) Flex Set, CBA Human Granzymeß Flex Set and CBA Human TNF Flex Set. Samples were measured using BD FACS Canto II or BD FACS Fortessa and analyzed using Diva software (BD Biosciences). Example 9 - Binding of T cell bispecific antibodies comprising optimized CD3 binders to CD3 and EGFRvIII and to recombinant CD3

Binding of EGFRvIII TCBs to recombinant CD3 was assessed by SPR using TCBs with optimized (EGFRvIII TCB CD3 _optimized ) or original (EGFRvIII TCB CD3 _original ) CD3 binding sequences, as described above for TYRP1 TCBs in Example 5. The capture antibody was coupled to the sensor wafer surface by direct immobilization at pH 5.0 at approximately 5200 resonance units (RU) using a standard amine coupling kit (GE Healthcare) and TCB molecules were captured at 20 nM at a flow rate of 10 μl 30 seconds.

The _KD values for binding to human and cynomolgus CD3 were determined to be 30 nM and 20 nM _optimized for TYRP1 TCB CD3, respectively, and were similar to the _original values of TYRP1 TCB CD3 (40 nM and 30 nM, respectively).

This shows that under stress-free conditions, the two TCBs comprising CD3- _optimized or CD3- _naive bind equally well to recombinant CD3.

Binding of EGFRvIII TCBs to recombinant human CD3 was also assessed after 14 days of temperature stress at 37°C or 40°C using TCBs with optimized or native CD3 binding sequences. The experiments were performed as described in Example 2 above, using TCB instead of IgG molecules.

The results of this experiment are shown in Figure 21.

As can be seen in Figure 21, the TCB containing the optimized CD3 binder CD3 _optimized showed significant binding to CD3 after stress (2 weeks at 37°C, pH 7.4) compared to the TCB containing the original CD3 binder CD3 _original Increase. This result again confirms that the improved properties of the optimized CD3 binder (see Example 2) are maintained at TCB levels. Binding to recombinant EGFRvIII

Binding of EGFRvIII TCB to recombinant EGFRvIII was assessed by SPR.

SPR experiments were performed on a Biacore T200 with HBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% (v/v) Surfactant P20 (GE Healthcare)).

Anti-Fc antibody (GE Healthcare) was coupled directly on the CM5 sensor wafer at pH 5.0 using a standard amine coupling kit (GE Healthcare). EGFRvIII TCB (5 nM) was captured at a flow rate of 10 µl/min for 30 s (see below). A 3-fold dilution series of EGFRvIII antigen was passed over a flow cell at 30 µl/min for 200 seconds and the associated phase was recorded. The dissociation phase was monitored for 300 seconds and triggered by changing the sample solution to HBS-EP. After each cycle, the wafer surface was regenerated using one injection of 3 M _MgCl2 at 20 μl/min for 30 seconds.

The antigen used contains the extracellular domain of human EGFRvIII fused to an Avi tag and a C-terminal His tag (EGFRvIII-ECD avi his; SEQ ID NO: 36).

Bulk refractive index differences were corrected by subtracting the responses obtained on the reference flow cell (not captured by the TCB). Affinity constants (K _D ) were derived from kinetic rate constants by fitting 1:1 Langmuir binding using BIAeval software (GE Healthcare). The apparent affinity constant, _KD , was approximately estimated for this 2:1 interaction by kinetic analysis via rate constants using a 1:1 binding fit.

The _KD value (affinity) for binding to human EGFRvIII was determined to be 6 nM for EGFRvIII TCBs comprising CD3 _optimized or CD3 _native .

Binding of EGFRvIII TCBs to recombinant EGFRvIII was also assessed after 14 days of temperature stress at 37°C or 40°C using TCBs with optimized or native CD3 binding sequences. Experiments were performed as described in Example 5 above, using EGFRvIII-ECD avi his as the antigen (see above).

The results of this experiment are shown in FIG. 22 . These results demonstrate that the binding of both TCBs to human EGFRvIII is not affected by stress conditions. Binds to CD3 on Jurkat cells

As described above in Example 2, binding of CD3 on the human reporter T cell line Jurkat NFAT was determined by FACS of EGFRvIII TCB comprising the optimized CD3 binder "CD3 _optimized " or the original CD3 binder "CD3 _original ".

As shown in Figure 23, TCB comprising the optimized CD3 binder "CD3 _optimized " and the original CD3 binder "CD3 _original " bound CD3 on Jurkat cells equally well. Binding to EGFRvIII on U87MG-EGFRvIII cells

Binding to EGFRvIII was determined by FACS on the human glioblastoma cell line U87MG-EGFRvIII against EGFRvIII TCBs comprising either the CD3- _optimized or CD3- _native EGFRvIII binder P063.056 or the CD3- _native EGFRvIII clone P056.021. Also included is the EGFRvIII binder P063.056 in IgG format.

As shown in Figure 24, all three TCBs bound with high affinity to EGFRvIII expressed on U87MG-EGFRvIII cells, and binding was not impaired by conversion to the TCB form. Example 10 - Functionally active CD3 activation of T cell bispecific antibodies comprising optimized CD3 binders

In the presence of EGFRvIII-positive glioblastoma cells DK-MG, U87MG-huEGFRvIII, and EGFRwt-positive MKN45 cells, assays containing selected EGFRvIII binders (P063.056) and optimized CD3 binding were tested in the Jurkat NFAT reporter cell assay Sub-CD3 _optimized or native CD3 binding sub-CD3 _native EGFRvIII TCB, as described above in Example 8.

As seen with IgG (Example 3), the two TCBs containing CD3- _optimized or CD3- _naive had similar functional activities on Jurkat NFAT reporter cells and included CD3 activation in a concentration-dependent manner (Figure 20). Target cell killing

The EGFRvIII TCB containing the selected EGFRvIII binder (P063.056) and the optimized CD3 binder CD3 _optimized or the original CD3 binder CD3 _original and the EGFRvIII TCB containing the parental EGFRvIII binder P056.021 and the CD3 binder CD3 _original in the presence of Comparisons were made in tumor cell killing experiments in the case of the glioblastoma cell line U87MG-EGFRvIII and PBMC, as described in Example 8 above. As seen on Jurkat NFAT reporter cells, the functional activities of TCB and CD3- _optimized or CD3- _naive were similar in inducing tumor cell lysis and CD4 and CD8 T cell activation, as measured by CD69 upregulation (Figure 25).

In addition, the functional activity of EGFRvIII TCB with EGFRvIII binder P063.056 and optimized CD3 binder CD3 _optimized in "2+1 format" (illustrated in Figure 6) was similar to that in "1+1 head-to-tail format" (schematically EGFRvIII TCBs with the same EGFRvIII and CD3 binders were compared (generically depicted in Figure 1G). These two EGFRvIII TCBs were tested in the Jurkat NFAT reporter cell assay and in the tumor killing assay with the glioblastoma cell line U87MG-EGFRvIII, as described in Example 8. EGFRvIII TCB in the 2+1 form has a significant role in CD3 activation as measured in the Jurkat NFAT reporter cell assay (Figure 26) and in inducing tumor cell killing and T cell activation in the killing assay with PBMC (Figure 27). Excellent functional activity. Example 11 - Functional Characterization of T Cell Bispecific Antibodies Comprising Optimized CD3 Binders T Cell Proliferation and Activation Using EGFRvIII TCB

The functional activity of EGFRvIII TCB containing selected EGFRvIII binder (P063.056) and optimized CD3 binder CD3 _optimized or original CD3 binder CD3 _original was compared with EGFRvIII containing parental EGFRvIII binder P056.021 and CD3 binder CD3 _original TCBs were compared in T cell proliferation assays on U87MG-EGFRvIII cells (Figure 28). All three TCB armies induced strong proliferation and activation of CD4 T cells and CD8 T cells. The P063.056 EGFRvIII TCB with CD3 _optimization was more active than the other two EGFRvIII TCBs. Tumor cell lysis with EGFRvIII TCB

Next, EGFRvIII TCBs containing the selected EGFR binders (P063.056) and optimized CD3 binders CD3- _optimized and EGFRvIII TCBs containing the parental EGFRvIII binders P056.021 and CD3 binders CD3 _original were used in tumor cell lysis assays. PBMCs co-cultured with DK-MG cells were tested (Figure 29). In this assay, in addition to tumor cell lysis, T cell activation was also measured, and cytokine release was measured as an additional readout. As seen previously, P063.056 EGFRvIII TCB with CD3 _optimization was more active than P056.021 EGFRvIII TCB with CD3 _native with respect to tumor cell lysis, T cell activation and release of IFNγ and TNFα. T cell activation and tumor cell lysis using TYRP1 TCB

The functional properties of TYRP1 TCB-induced cytokine release were tested by co-culturing primary melanoma cell line M150543 with PBMC isolated from healthy donors. Tumor cell lysis mediated by TYRP1 TCB using T cells was analyzed after 24 hours and 48 hours of treatment (Figure 30). After 48 hours of treatment, IFNy and TNFa release into supernatant and CD4 and CD8 T cell activation were analyzed. TYRP1 TCB was able to induce efficient tumor cell lysis after 24 hours. This was accompanied by strong activation of CD4 and CD8 T cells as determined by positive regulation by CD25 and massive release of IFNy and TNFa. Method PBMC isolation

Peripheral blood mononuclear cells (PBMC) were prepared by Histopaque density centrifugation of fresh blood obtained from healthy human donors. Fresh blood was diluted with sterile PBS and layered in a Histopaque gradient (Sigma, #H8889). After centrifugation (450 xg, 30 min, room temperature), the plasma above the PBMC-containing mesophase was discarded and the PBMCs were transferred to new falcon tubes, which were subsequently filled with 50 ml PBS. The mixture was centrifuged (400 xg, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was washed twice with sterile PBS (centrifugation step 350 xg, 10 min). The resulting PBMC populations were automatically counted (ViCell) and stored in RPMI1640 medium in a cell incubator at 37°C, 5% _CO2 until further use (no more than 24 hours) or frozen and stored in liquid nitrogen until further Used, the medium contains 10% FCS and 1% GlutaMAX. Frozen PBMCs were thawed the day before use and incubated overnight in medium at 37°C. T cell proliferation

Briefly, target cells were collected, counted, and washed twice with PBS. Cells were resuspended in PBS at 5 million cells/mL. Cells were stained with the cell proliferation dye eFluor 670 (eBioscience, #65-0840-85) at a final concentration of 5 µM for 10 minutes at 37°C. To stop the staining reaction, 4 volumes of cold complete cell medium were added to the cell suspension and incubated at 4°C for 5 minutes, and then washed 3 times with medium. Labeled target cells were counted in RPMI1640, 10% FCS and 1% GlutaMax and adjusted to 100,000 cells per ml. 10'000 target cells per well were seeded into 96-well plates. Treatments were then added at the indicated concentrations, and finally 100'000 PBMCs isolated from healthy donors were added per well. Cells were incubated at 37°C for 5 days, then PBMCs were harvested and treated with CD3 BUV395 (BioLegend, #563548), CD4 PE (BioLegend, #300508), CD8 APC (BioLegend, #344722), CD25 PE/Cy7 (BioLegend, #302612) dyeing. Proliferation was determined by measuring dilution of eFluor 670 dye in CD4 T cells and CD8 T cells by flow cytometry (FACS Fortessa, BD Bioscience) and by measuring CD25 upregulation of activated CD4 and CD8 T cells. T cell-mediated tumor cell killing

Target cells were harvested with trypsin/EDTA, washed, and plated at a density of 30,000 cells/well using flat-bottomed 96-well dishes. Cells were allowed to adhere overnight. For poisoning assays, antibodies (triplicates) were added at the indicated concentrations. PBMCs were added to target cells to obtain a final effector to target (E:T) ratio of 10:1. Assessed by quantification of LDH released from apoptotic/necrotic cells into the supernatant of cells (LDH Detection Kit, Roche Applied Science, #11 644 793 001) after 24 hours of incubation at 37°C, 5% CO ₂ target cell killing. Maximal lysis of target cells (= 100%) was achieved by incubating target cells with 1% Triton X-100. Minimal lysis (= 0%) refers to target cells co-incubated with effector cells without the bispecific construct. T cell activation

Activation of CD8 and CD4 T cells following TCB-mediated T cell killing of target cells was assessed by flow cytometry using antibodies recognizing the T cell activation markers CD25 (late activation marker) and CD69 (early activation marker). After 48 hours of incubation, PBMCs were transferred to round bottom 96-well plates, centrifuged at 350 xg for 5 minutes, and washed twice with FACS buffer. Surface staining for CD4 APC (BioLegend, #300514), CD8 FITC (#344704, BioLegend), CD25 BV421 (BioLegend, #302630) and CD69 PE (BioLegend, #310906) was performed according to the supplier's instructions. Cells were washed twice with 150 μl/well FACS buffer and fixed with 100 μl/well Fixation Buffer (BD, #554655) for 15 minutes at 4°C. After centrifugation, samples were resuspended in 200 μl/well FACS buffer. Samples were analyzed with BD FACS Fortessa. cytokine secretion

Cytokinetic secretion in supernatants was measured by flow cytometry using a Cytometry Bead Array (CBA) according to the manufacturer's instructions, but only 25 μl supernatant and beads were used instead of 50 μl beads and sample. The following CBA sets (BD Biosciences) were used: CBA Human Interferon Gamma (IFNγ) Flex Set and CBA Human TNF Flex Set. Samples were measured using BD FACS Canto II or BD FACS Fortessa and analyzed using Diva software (BD Biosciences). Example 13 - In Vitro Efficacy of T Cell Bispecific Antibodies Comprising Optimized CD3 Binders

The antitumor efficacy of TYRP1 TCB (comprising the optimized CD3 binder identified in Example 1) was tested in an xenograft mouse model of human tumor cell lines, an IGR-1 melanoma xenograft model.

IGR-1 cells (human melanoma) were cultured in DMEM medium containing 10% FCS (Sigma). Cells were cultured at 37°C in a water-saturated atmosphere with 5% CO ₂ . Use channel 6 for transplantation. The cell viability was 96.7%. Mice were injected subcutaneously with ^2x106 cells per animal in 100 μl of RPMI cell culture medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences).

Fully humanized NSG female mice (Roche Glycart AG, Switzerland) were maintained under specific pathogen-free conditions according to committed instructions (GV-Solas; Felasa; TierschG) on a daily cycle of 12 h light/12 h dark. The experimental research protocol has been reviewed and approved by the local government (ZH223/2017). Regular ongoing health monitoring.

On study day 0, mice were injected subcutaneously with ^2x106 IGR-1 cells and randomly weighed. Twenty days after tumor cell injection (tumor volume > 200 mm ³ ), mice were intravenously injected with 10 μg (0.5 mg/kg) TYRP1 TCB twice weekly for five weeks. All mice were intravenously injected with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer, and mice in the treatment group were injected with the TYRP1 TCB construct. If necessary, to obtain the appropriate amount of antibody per 200 μl, the stock solution can be diluted with histidine buffer. Tumor size was measured 3 times per week with calipers and plotted as volume in mm ³ +/- SEM using GrahPad Prism software. Statistical analysis was performed with JMP12 software.

Figure 31 shows that TYRP1 TCB mediates significant efficacy in inhibiting tumor growth compared to the vehicle (68% TGI, p=0.0058*) group.

The antitumor efficacy of EGFRvIII TCB (comprising the optimized CD3 binder identified in Example 1) was also tested in an xenograft mouse model of human tumor cell lines, the U87-EGFRvIII glioblastoma xenograft model.

U87 cells (human glioblastoma) were originally obtained from ATCC (Manassas, USA) and stably transfected to express human EGFRvIII protein (Roche Glycart AG, Switzerland). After expression, cells are deposited in the Roche Glycart in-house cell bank. U87-EGFRvIII cell line was cultured in DMEM medium containing 10% FCS (Sigma) and 0.5 µg/ml puromycin (Invitrogen). Cells were cultured at 37°C in a water-saturated atmosphere with 5% CO ₂ . Use channel 8 for migration. The cell viability was 94.7%. Mice were injected subcutaneously with 5x10 ⁵ cells per animal in 100 μl of RPMI cell culture medium (Gibco) using a 1 ml tuberculin syringe (BD Biosciences).

Fully humanized NSG female mice (Roche Glycart AG, Switzerland) were maintained under specific pathogen-free conditions according to committed instructions (GV-Solas; Felasa; TierschG) on a daily cycle of 12 h light/12 h dark. The experimental research protocol has been reviewed and approved by the local government (ZH223/2017). Regular ongoing health monitoring.

On study day 0, mice were injected subcutaneously with ^5x105 U87-EGFRvIII cells and randomly weighed. Two weeks after tumor cell injection (tumor volume > 200 mm ³ ), mice were intravenously injected with 10 μg (0.5 mg/kg) of EGFRvIII TCB twice weekly for three weeks. All mice were intravenously injected with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer, and mice in the treatment group were injected with the EGFRvIII TCB construct. If necessary, to obtain the appropriate amount of antibody per 200 μl, the stock solution can be diluted with histidine buffer. Tumor size was measured 3 times per week with calipers and plotted as volume in mm ³ +/- SEM using GrahPad Prism software.

Figure 32 shows that EGFRvIII TCB mediated significant efficacy in inhibiting tumor growth, with all mice achieving complete remission. Example 14 - PK study with EGFRvIII TCB in mice

The pharmacokinetics of CD3- _optimized EGFRvIII TCBs comprising the optimized CD3 binder were investigated following 1 mg/kg intravenous bolus administration to human FcRn transgenic mice (line 32, homozygous) and NOD-SCID mice ( PK). Serial blood microsamples were obtained from human FcRn transgenic (tg) mice and NOD-SCID mice up to 672 hours (9 samples per mouse from 5 minutes to 672 hours post-dose). Mouse serum samples treated with EGFRvIII TCB were assayed using a specific enzyme-binding immunosorbent assay (ELISA) under non-GLP conditions. EGFRvIII TCBs were captured on avidin-coated microtiter plates (SA-MTP) using biotinylated EGFRvIII antigen (huEGFRvIII his biotin). Bound EGFRvIII TCB was detected with a digoxigenin-labeled monoclonal antibody against human IgG1 Fc (PGLALA) (see Example 3), followed by the addition of anti-digoxigenin-POD secondary Detection antibodies. The signal is generated by adding a peroxidase substrate (ABTS). The calibration range is 2.35 ng/ml to 150 ng/ml, with 2.5 ng/ml being the lower limit of quantitation (LLOQ).

The results of this study are shown in Table 7. The PK profile of the EGFRvIII TCB was within the expected range for both tested mouse strains. This indicates that engineering of the CDRs of the CD3 binder does not give rise to additional sequence responsibilities that would affect antibody clearance. Table 7. Clearance data for huFcRn tg mice and NOD-SCID mice (ml/day/kg; mean). mouse strain EGFRvIII TCB (CD3 _optimized ) hFcRn tg32 12.4 NOD-SCID 10.1 Example 15 - FOLR1 TCB with CD3 _Optimization as CD3 Binder : Transformation and Generation of FOLR1 TCB with CD3 _Optimization as CD3 Binder

To generate CD3 _optimized FOLR1 TCBs ("canonical 2+1 format" = CD3 Fab outside the Fc knob, FOLR1 Fab inside, Figure 33A, SEQ ID NOs: 127, 128, 129), the CD3 binder CD3 _optimized The variable domains are inserted into a suitable expression vector. The molecule consists of a human IgG1 framework with mutations in the Fc region (LL234/235AA and P329G) to eliminate Fc effector functions. The T cell bispecific molecule is produced as a proprietary 2+1 heterodimer based on the knob-hole technology (two binding moieties for the target antigen and one for CD3). Transfection and expression of FOLR1 TCB with CD3 _optimized as CD3 binder

Expression of FOLR1 TCB was performed by transient transfection of ExpiCHO-S™ cells (ExpiCHO™ Expression System; Thermo/Gibco #A29133). ExpiCHO-S cells were pre-cultured according to the manufacturer's instructions. On the day of transfection, seed 500 ml of cells at 6 × 10E6 viable cells/ml into disposable sterile shaker flasks. For optimal transfection, use a total of 1.0 μg of plastid DNA and 0.64 μl of ExpiFectamine™ CHO Reagent per ml of culture volume.

Individual dilutions of ExpiFectamine™ CHO reagent in cold OptiPRO™ medium and DNA were incubated in cold OptiPRO™ medium for 5 minutes at room temperature. Add the diluted ExpiFectamine™ CHO Reagent to the diluted DNA, mix well and incubate for an additional 5 minutes at room temperature. Subsequently, the complex mixture was added to the cells and incubated on an orbital shaking platform in a 37°C incubator with ≥80% relative humidity and 8% CO2.

A high titer protocol was used for protein expression according to the supplier's recommendation: Add ExpiFectamine™ CHO Enhancer and a single feed on day 1 post-transfection; cells were warmed to 32°C on day 1 post-transfection. On day 8 post-transfection, cell supernatants were collected for purification. Purification of FOLR1 TCB

FOLR1 TCB was purified by protein A affinity chromatography, followed by ion exchange chromatography and particle size sieve chromatography. Briefly, the supernatant was loaded onto a HiTrap MabSelect SuRe column (GE Healthcare) equilibrated with 1 x PBS pH 7.4. After a washing step with 5 column volumes of equilibration buffer, proTCB was eluted with 100 mM sodium acetate pH 3.0. The flow rate was set at 5 ml/min. Pooled fractions were diluted with water (1:5 v/v) and loaded onto POROS HS 50 columns (ThermoFisher Scientific) equilibrated with 40 mM sodium acetate pH 5.5. After washing with equilibration buffer, TCB was eluted using a 40 mM to 1 M sodium acetate gradient over 27 column volumes. The flow rate was set at 7 ml/min. The collected fractions were analyzed by analytical particle size sieve chromatography (Waters BioSuite) and pooled according to the content of monomeric species. Subsequently, the pooled fractions were concentrated to a final volume of 15 ml using an Amicon Ultra device (Millipore). The concentration cell was loaded on a HiLoad 26/60 Superdex preparative grade column (GE Healthcare) with a column volume of 320 ml. The running buffer was 20 mM histidine-HCl, 140 mM NaCl pH 6.0, and the flow rate was set at 3 ml/min. Fractions were pooled according to the content of monomeric species. molecular Pure Line ID Yield [mg/L] Product peak SEC [%] Main peak CE-SDS [%] Endotoxin [EU/ml] FOLR1 TCB_cl22 P1AF1257 58.8 98.5 96.9 < 0.2 Table 8 : Production/purification results of FOLR1 TCB using cl22 as CD3 binder Example 16 - FOLR1-TCB (CD3 _optimized ) mediated Jurkat NFAT activation

FOLR1-TCB containing the CD3 clone 22 binder was first tested in the Jurkat NFAT reporter assay.

FOLR1-targeting T cell bispecific antibody (TCB) binds to both huFOLR1-coated beads and CD3ε (Jurkat NFAT) on T cells, thereby inducing T cell activation. Since Jurkat NFAT luciferase cells express luciferase upon activation via CD3epsilon (CD3ε), T cell activation can be measured as luminescence. The Jurkat-NFAT reporter cell line (Promega) is a human acute lymphoblastic leukemia reporter cell line with the NFAT promoter expressing human CD3ε. If TCB binds tumor targets and CD3 (cross-linked) binds CD3ε luciferase performance can be measured in luminescence after addition of One-Glo substrate (Promega).

Jurkat NFAT assay medium: RPMI1640, 2 g/l glucose, 2 g/l NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1 × NEAA, 1 × sodium pyruvate

Jurkat NFAT medium: RPMI1640, 2 g/l glucose, 2 g/l NaHCO3, 10% FCS, 25 mM HEPES, 2 mM L-glutamine, 1 × NEAA, 1 × sodium pyruvate; newly added hygromycin B 200 µg/ml.

65 μl Avidin Dynabead diluted in 10 ml PBS. The beads were centrifuged at 400rcf for 4 minutes and the supernatant was removed. 30 μg of biotinylated FolR1 antigen was then added to 1.5 ml of DPBS, and then to avidin beads. The bead antigen mixture was incubated for 1 hour at 4°C with slow rotation. After incubation, 10 ml of DPBS was added to the bead-ag conjugate, centrifuged (4 min, 400 rcf), and the supernatant discarded. The conjugate was washed again with 5 ml DPBS and the pellet was then resuspended in 6 ml assay medium. Effector cells (Jurkat NFAT) were harvested, counted and checked for viability. Cells were centrifuged at 350 rcf for 4 min, then resuspended in 12 ml assay medium. Add cAMP to the effector cell suspension (2% final volume).

Coated beads (in 10 μl/well) and Jurkat NFAT effector cells with cAMP (20.000 cells/well in 20 μl/well) were mixed and added to a 384-well white-walled clear dish (Greiner BioOne). FOLR1-TCB was diluted in jurkat assay medium before preparing dilution columns and 10 μl was added per well. Cells were incubated with beads and TCB/medium for 5.5 hours at 37°C in a humidified incubator, then removed from the incubator for about 10 minutes to acclimate to room temperature, then used in Tecan Spark with detection time 0.5 sec/well read luminescence.

FOLR1-TCB induced dose-dependent Jurkat NFAT activation using huFOLR1-coated beads for cross-linking (Figure 34). Example 17 - T cell killing mediated by FOLR1 pro - TCB (CD3 clone 22)

To assess the efficacy of FOLR1-TCB with CD3 _optimization , FOLR1-positive Ovcar-3 cells were used to assess target cytotoxicity and T cell activation mediated by FOLR1-TCB. Human PBMCs were used as effector cells, and T cell activation markers were stained after 48 hours of incubation with molecules and cells. Human peripheral blood mononuclear cells (PBMC) were isolated from skin-colored hemospheres obtained from healthy human donors. The skin color hemocytometer was diluted 1:1 with sterile PBS and layered with a Histopaque gradient (Sigma, #H8889). After centrifugation (450 xg, 30 min, no interruptions, room temperature), the PBMC-containing mesophase was transferred to a new falcon tube, which was then filled with 50 ml of PBS. The mixture was centrifuged (400 xg, 10 min, room temperature), the supernatant was discarded, and the PBMC pellet was resuspended in 2 ml ACK buffer for erythrocyte lysis. After incubation at 37°C for about 2 to 3 minutes, tubes were filled to 50 ml with sterile PBS and centrifuged at 350 xg for 10 minutes. This washing step was repeated once before resuspending the PBMCs in Advanced RPMI1640 medium containing 2% FCS, 1X GlutaMax and 10% DMSO. PBMCs were slowly frozen in CoolCell® cell freezing containers (BioCision) at -80°C and then transferred to liquid nitrogen. Adherent target cells were harvested with trypsin/EDTA, counted, checked for viability, and resuspended in assay medium (RPMI1640, 2% FCS, 1X GlutaMax) the day before the start of the assay. PBMCs were thawed in RPMI1640 medium (10% FCS, 1X GlutaMax) approximately 24 hours before the start of the assay. PBMCs were centrifuged at 350 g for 7 minutes and resuspended in fresh medium (RPMI1640, 10% FCS, IX GlutaMax). PBMC were retained for up to 24 hours before being used for analysis. Assay medium was RPMI + 2% FCS + 1% GlutaMax.

Target cells were seeded at a density of 20 000 cells/well (50 μl/well in assay medium) using a 96-well plate. Cells were incubated overnight at 37°C in a humidified incubator. Molecules were diluted in assay medium and added in triplicate at the indicated concentrations. PBMCS were harvested and centrifuged at 350 g for 7 minutes before resuspending in assay medium. Add 200,000 huPBMCs, 100 μl per well (E:T 10:1, based on the number of target cells seeded) before incubating the plate at 37°C for 48 hours. Assessed by quantifying LDH released from apoptotic/necrotic cells into cell supernatant after 48 hours of incubation at 37°C, 5% CO2 (LDH Detection Kit, Roche Applied Science, #11 644 793 001) target cell killing. Maximal lysis of target cells (= 100%) was achieved by incubating target cells with 1% Triton X-100 for 1 hour prior to LDH readout. Minimal lysis (= 0%) refers to target cells co-incubated with effector cells without any TCB. LDH release was measured by measuring absorbance (A492nm-A650nm).

T cell activation was assessed by quantifying CD69 on CD4-positive and CD8-positive T cells after 48 hours of incubation at 37°C, 5% CO2.

DPBS was added to the wells containing PBMCs before centrifuging the plates at 400 x g for 4 min. The supernatant was aspirated and the cells were washed again with PBS, centrifuged, the supernatant was removed, and the cell pellet was resuspended by carefully vortexing the plate. 5 μl LIVE/DEAD™ Fixable Aqua Dead Cell Stain diluted 1:1000 in DPBS. Add 50 μl of diluted dye to wells containing PBMCs. An empty well was prepared by adding 1 drop of compensation beads (Invitrogen ArcTM, reactive beads) and 1 μl of undiluted LIVE/DEAD dye was added. Incubate the plate at 4°C for 30 minutes. To remove excess color, cells were washed twice, first with 150 μl PBS and second with 150 μl FACS buffer. Centrifuge the dish at 400 x g for 4 min. The supernatant was removed and cells were resuspended by careful vortexing. Add 1 drop of negative beads (Invitrogen ArcTM, Negative Beads) to compensation control wells containing LIVE-stained beads. Add 25 μl of diluted CD4/CD8/CD25/CD69 antibody mix (0.8 μl/well of each color), single antibody (for compensation control; 0.8 μl AB + 24 μl FACS buffer) or FACS buffer (for unstained Control) were added to the resuspended cells to the wells, and the plate was incubated at 4°C for 60 minutes. To remove unbound antibody, cells were washed twice with 150 μl of FACS buffer per well. The supernatant was removed after centrifugation and the cells were resuspended by careful vortexing. Cells were fixed overnight in 150 ul FACS + 1 % PFA buffer for analysis the following week. Cells were resuspended in 150 μl FACS buffer the next day. Fluorescence was measured using the FACS LSR Fortessa.

Dose-dependent target cell killing was shown for Ovcar-3 cells incubated with huPBMC and FOLR1-TCB (CD3 _optimized ) (FIG. 35A). Dashed lines show huPBMCs incubated with target cells, but without TCB. For CD4 and CD8 positive T cells, target cytotoxicity correlated with T cell activation as measured by CD69 (Figure 35B). * * *

Although the foregoing invention has been described in detail by way of illustration and example for clarity of understanding, such description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Figure 1. Exemplary configuration of a (multispecific) antibody of the invention. (A, D) Schematic representation of the "1+1 CrossMab" molecule. (B, E) Schematic representation of the "2+1 IgG Crossfab" molecule with sequential replacement ("inverted") of the Crossfab and Fab compositions. (C, F) Schematic representation of the "2+1 IgG Crossfab" molecule. (G, K) Schematic representation of the "1+1 IgG Crossfab" molecule with sequential replacement ("inversion") of the Crossfab and Fab compositions. (H, L) Schematic representation of the "1+1 IgG Crossfab" molecule. (I, M) Schematic representation of the "2+1 IgG Crossfab" molecule with two CrossFabs. (J, N) Schematic representation of a "2+1 IgG Crossfab" molecule with two CrossFabs and a sequence of Crossfab and Fab compositions replaced ("inverted"). (O, S) Schematic representation of the "Fab-Crossfab" molecule. (P, T) Schematic representation of the "Crossfab-Fab" molecule. (Q, U) Schematic representation of the "(Fab) ₂ -Crossfab" molecule. (R, V) Schematic representation of the "Crossfab-(Fab) ₂ " molecule. (W, Y) Schematic representation of the "Fab-(Crossfab) ₂ " molecule. (X, Z) Schematic representation of the "(Crossfab) ₂ -Fab" molecule. Black dots: Optional modifications in the Fc domain that promote heterodimerization. ++, --: amino acids of opposite charges are optionally introduced into the CH1 and CL domains. Crossfab molecules are described as comprising the exchange of VH and VL domains, but may (in the aspect in which no charge modifications are introduced in the CH1 and CL domains) alternately comprise the exchange of CH1 and CL domains. Figure 2. Relative binding activities of original and optimized CD3 binders CD3 _original and CD3 _optimized and recombinant CD3 by SPR under stress-free conditions after 14 days at 40°C pH 6 or 14 days at 37°C pH 7.4 Post-measurement (IgG format). Figure 3. Native and optimized CD3 binders CD3 _native and CD3 _optimized binding to Jurkat NFAT cells as measured by flow cytometry (IgG format). Antibodies bound to Jurkat NFAT cells were detected by a fluorescently labeled anti-human Fc-specific secondary antibody. Figure 4. Schematic representation of the CD3 activation assay used in Example 3. Figure 5. Jurkat NFAT activation (IgG format) with original and optimized CD3 binders CD3 _original and CD3 _optimized . Jurkat NFAT reporter cells were co-incubated with anti-PGLALA expressing CHO (CHO-PGLALA) cells in the presence of CD3 _native or CD3 _optimized IgG PGLALA or CD3 _optimized IgG wt (as negative controls). CD3 activation was quantified by measuring luminescence after 24 hours. Figure 6. Schematic representation of T cell bispecific antibody (TCB) molecules prepared in the Examples. All TCB antibody molecules tested were produced as "2+1 IgG CrossFab, inverted" with charge modifications (VH/VL exchange in CD3 binder, charge modification in target antigen binder, EE = 147E, 213E ; RK = 123R, 124K). Figure 7. Relative binding activity of TYRP1 TCBs comprising native or optimized CD3 binders CD3 _native or CD3 _optimized and recombinant CD3 by SPR under stress-free conditions after 14 days at 40°C pH 6 or at 37°C pH 7.4 Measure after 14 days. Figure 8. Relative binding activity of TYRP1 TCBs comprising native or optimized CD3 binders CD3 _native or CD3 _optimized or corresponding to TYRP1 IgG and recombinant TYRP1 by SPR under stress-free conditions at 40°C pH 6 after 14 days or Measured after 14 days at 37°C pH 7.4. Figure 9. Binding of TYRP1 TCB comprising naive or optimized CD3 binders CD3 _naive or CD3 _optimized to Jurkat NFAT cells as measured by flow cytometry. TCB bound to Jurkat NFAT cells was detected by a fluorescently labeled anti-human Fc-specific secondary antibody. Figure 10. Jurkat NFAT activation with TYRP1 TCB containing original or optimized CD3 binders. Jurkat NFAT reporter cells were co-cultured with the melanoma cell line M150543 in the presence of TYRP1 TCB CD3 _original or TYRP1 TCB CD3 _optimized . CD3 activation in the presence of TCB was quantified by measuring luminescence after 24 hours. Figure 11. Tumor cell killing and T cell activation with TYRP1 TCBs containing native or optimized CD3 binders. Intoxication of melanoma cell line M150543 following TYRP1 TCB CD3 _original and TYRP1 TCB CD3 _optimized treatment of PBMCs from three different healthy donors (AF: Donor 1, GL: Donor 2, MR: Donor 3) Determined by LDH release after 24 hours (A, G, M) and 48 hours (B, H, N). At the same time, CD8 (E, F, K, L, Q, R) and CD4 (C, D, I, J, O, P) T cells on CD25 (C, E, I, K, O, Q) and CD69 (D, F, J, L, P, R) were measured by flow cytometry as markers of T cell activation after 48 hours. Figure 12. Specific binding of EGFRvIII IgG PGLALA. Specific binding of EGFRvIII IgG PGLALA antibody to EGFRvIII non-cross-reactive to EGFRwt expressed by flow cytometry in CHO-EGFRvIII (A), EGFRvIII positive DK-MG (B) and EGFRwt MKN-45 (C) test on. Cetuximab was included as a positive control for EGFRwt expression. Figure 13. CAR J activation with EGFRvIII IgG PGLALA. Jurkat NFAT reporter cells expressing anti-PGLALA CAR were co-incubated with EGFRvIII expressing DK-MG cells and EGFRvIII IgG PGLALA antibody or DP47 IgG PGLALA (as negative controls). Jurkat NFAT cell activation was quantified by measuring luminescence after 22 h. Figure 14. Binding of EGFRvIII IgG PGLALA and corresponding TCB to EGFRvIII. Specific binding to CHO-EGFRvIII (A) and MKN-45 (B) cells as IgG PGLALA and EGFRvIII binder converted to TCB was measured by flow cytometry. Figure 15. Jurkat NFAT activation with EGFRvIII TCB. Jurkat NFAT activation was determined as a marker of CD3 engagement with EGFRvIII TCB in the presence of EGFRvIII positive DK-MG cells. DP47 TCB was included as a negative control. Figure 16. Tumor cell lysis with EGFRvIII TCB. After co-culture with freshly isolated PBMC and EGFRvIII-positive DK-MG cells (A, B) or EGFRwt-positive MKN-45 cells (C, D) for 24 hours (A, C) or 48 hours (B, D) Afterwards, EGFRvIII TCB was determined to induce lysis of specific tumor cells. Figure 17. T cell activation with EGFRvIII TCB. Activation markers CD25 (A, C, E, G) or CD69 (B, D, F, H) on CD4 T cells (AD) or CD8 T cells (EH) with freshly isolated PBMC and EGFRvIII positive DK EGFRvIII TCB-induced T cell activation was determined after co-culture with MG cells (A, B, E, F) or EGFRwt positive MKN-45 cells (C, D, G, H). Figure 18. Cytokine release with EGFRvIII TCB. After co-culture with freshly isolated PBMC and EGFRvIII positive DK-MG cells (AC) or EGFRwt positive MKN-45 cells (DF), the induction of IFNγ (A, D), TNFα (B, E) and particles by EGFRvIII TCB was determined Release of enzymes B (C, F). Figure 19. Specific binding of affinity matured EGFRvIII IgG PGLALA. The specific binding of affinity matured EGFRvIII antibody to EGFRvIII on U87MG-EGFRvIII cells (A) and EGFRwt positive cell line MKN-45 (B) was compared to the parental EGFRvIII binder. Figure 20. Jurkat NFAT activation with EGFRvIII TCB. Jurkat NFAT activation was determined as a marker of CD3 engagement with EGFRvIII TCB in the presence of EGFRvIII positive DK-MG cells (A), U87MG-EGFRvIII cells (B) and MKN-45 cells (C). DP47 TCB was included as a negative control. Figure 21. Relative binding activity of EGFRvIII TCB comprising native or optimized CD3 binder CD3 _native or CD3 _optimized to recombinant CD3 by SPR under stress-free conditions at 40°C pH 6 after 14 days or at 37°C pH 7.4 Measure after 14 days. Figure 22. Relative binding activity of EGFRvIII TCBs comprising native or optimized CD3 binders CD3 _native or CD3 _optimized to recombinant EGFRvIII by SPR under stress-free conditions after 14 days at 40°C pH 6 or at 37°C pH 7.4 Measure after 14 days. Figure 23. Binding of EGFRvIII TCBs comprising naive or optimized CD3 binders CD3 _naive or CD3 _optimized to Jurkat NFAT cells as measured by flow cytometry. TCB bound to Jurkat NFAT cells was detected by a fluorescently labeled anti-human Fc-specific secondary antibody. Figure 24. Binding of EGFRvIII TCBs comprising P063.056 or P056.021 EGFRvIII binders to U87MG-EGFRvIII cells was measured by flow cytometry. TCB bound to U87MG-EGFRvIII was detected by a fluorescently labeled anti-human Fc-specific secondary antibody. Figure 25. Tumor cell lysis and T cell activation with EGFRvIII TCB. EGFRvIII TCBs were determined to induce specific tumor cell lysis (A, B) and T cell activation ( C, D). DP47 TCB was included as a negative control. Figure 26. Comparison of Jurkat NFAT activation in EGFRvIII TCB 2+1 and 1+1 forms. Jurkat NFAT activation was determined as a marker of CD3 engagement with EGFRvIII TCB in 2+1 inverted form and 1+1 head-to-tail form in the presence of EGFRvIII positive U87MG-EGFRvIII cells. Figure 27. Comparison of tumor cell lysis and T cell activation of EGFRvIII TCB 2+1 and 1+1 forms. After co-culture with freshly isolated PBMC and U87MG-EGFRvIII cells for 24 hours (A, C) or 48 hours (B, D), EGFRvIII TCBs in 2+1 inverted form and 1+1 head-to-tail form were determined Induces lysis of specific tumor cells (A, B) and T cell activation (C, D). Figure 28. T cell activation and proliferation with EGFRvIII TCB. After co-culturing U87MG-EGFRvIII and PBMC isolated from healthy donors, it was determined that EGFRvIII TCB induced T cell proliferation (A, C) T cell activation of CD4 T cells (A, B) and CD8 T cells (C, D). Figure 29. Tumor cell lysis, T cell activation and cytokine release with EGFRvIII TCB. After co-culture of U87MG-EGFRvIII cells with PBMCs, it was determined that EGFRvIII TCB induced tumor cell lysis (A, B), T cell activation (C, D) and IFNγ and TNFα release. Tumor cell lysis was measured after 24 hours and 48 hours of treatment, and T cell activation and cytokine release were measured after 48 hours. Figure 30. Tumor cell lysis, T cell activation and cytokine release with TYRP-1 TCB. TYRP-1 TCBs were determined to induce tumor cell lysis (A, B), T cell activation (C, D) and IFNγ and TNFα (E, F) release after co-culture of patient-derived melanoma cell line M150543 with PBMCs . Tumor cell lysis was measured after 24 hours and 48 hours of treatment, and T cell activation and cytokine release were measured after 48 hours. Figure 31. In vivo efficacy of TYRP-1 TCB. The IGR-1 human melanoma cell line was injected subcutaneously into humanized NSG mice to study tumor growth inhibition in a subcutaneous xenograft model of melanoma. Significant tumor growth inhibition (TGI) was observed in the TYRP-1 TCB group (68% TGI, p=0.0058*) compared to the vehicle group. Figure 32. In vivo efficacy of EGFRvIII TCB. The U87-huEGFRvIII human glioblastoma cell line was injected subcutaneously into humanized NSG mice to study tumor growth inhibition in a subcutaneous xenograft model of glioblastoma cells. Significant tumor control was observed in the EGFRvIII TCB group, where all mice achieved complete remission. Figure 33. Form of the FolR1 TCB molecule. Figure 33A: A typical 2+1 TCB molecule with a CD3 Fab fused via a (G4S)2 linker to the VH of the internal FOLR1 Fab. Heterodimerization by Knob-Hole technique, PGLALA mutation in Fc. Figure 33B: Inverted 2+1 FOLR1 TCB with CD3 _optimized Fab inside the Fc knob. Figure 33C. A typical 1+1 head-to-tail FOLR1 TCB molecule in which a CD3 _optimized Fab is fused to a VH within an internal FOLR1 Fab via a (G4S)2 linker. Heterodimerization by Knob-Hole technique, PGLALA mutation in Fc. Figure 33D. Inverted 1+1 head-to-tail FOLR1 TCB with CD3 _optimized Fab inside the Fc knob. Figure 33E: 1+1 IgG-like FOLR1 TCB molecule with CD3 _optimized Fab on the Fc knob chain and FOLR1 Fab on the Fc hole chain. Fab. Heterodimerization by Knob-Hole technology, PGLALA mutation in Fc. Figure 34. FOLR1-TCB (CD3 _optimized ) mediated activation of Jurkat NFAT. Demonstrates FOLR1-TCB and CD3 _optimization -mediated activation of Jurkat NFAT. FOLR1-TCB was incubated with huFOLR1-coated beads and Jurkat NFAT effector cells for 5.5 h at 37°C. Dotted lines represent beads with Jurkat cells without TCB. Each point represents the mean of technical triplicates. Standard deviations are represented by error bars (n=1). Figure 35. Tumor cell lysis and T cell activation with FOLR1 (pro-) TCB (CD3 _optimized ). Dose-dependent tumor cell lysis and T cell activation were analyzed after co-incubation of human PBMC (effector cells) and FOLR1-positive target cells (Ovcar-3) with FOLR1 TCB for 24 and 48 hours (E:T = 10:1 , the effector is human PBMC). Tumor cell lysis was induced after 24 hours and 48 hours (A and B). T cell activation was measured by quantification of CD69 on CD4 and CD8 T cells by FACS after 48 hours of treatment. CD69-positive CD4 T cells (C) and CD8 T cells (D) are shown in the upper panel. In the lower panel, the median CD69{PE} is plotted against CD4 (E) and CD8 (F) positive T cells. Each point represents the mean of technical triplicates. Standard deviations are represented by error bars.

         <![CDATA[ <110> F. Hoffmann-La Roche AG]]>
           <![CDATA[ <120> Antibodies that bind to CD3 and FolR1]]>
           <![CDATA[ <130> P36031]]>
           <![CDATA[ <140>TW 110122163]]>
           <![CDATA[ <141> 2021-06-17]]>
           <![CDATA[ <150> EP 20181022.3]]>
           <![CDATA[ <151> 2020-06-19]]>
           <![CDATA[ <160> 137 ]]>
           <![CDATA[ <170> PatentIn v3.5]]>
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          Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr
                      100 105 110
          Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
                  115 120 125
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
              130 135 140
          Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
          145 150 155 160
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
                          165 170 175
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
                      180 185 190
          Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
                  195 200 205
          Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
              210 215 220
          Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
          225 230 235 240
          Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
                          245 250 255
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
                      260 265 270
          Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                  275 280 285
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
              290 295 300
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
          305 310 315 320
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
                          325 330 335
          Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
                      340 345 350
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
                  355 360 365
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
              370 375 380
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
          385 390 395 400
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
                          405 410 415
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
                      420 425 430
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                  435 440 445
          Leu Ser Leu Ser Pro
              450
           <![CDATA[ <210> 14]]>
           <![CDATA[ <211> 216]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CD3 original / CD3 optimized IgG LC]]>
           <![CDATA[ <400> 14]]>
          Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly
          1 5 10 15
          Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser
                      20 25 30
          Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly
                  35 40 45
          Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
              50 55 60
          Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala
          65 70 75 80
          Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
                          85 90 95
          Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg Thr Val
                      100 105 110
          Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
                  115 120 125
          Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
              130 135 140
          Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
          145 150 155 160
          Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
                          165 170 175
          Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
                      180 185 190
          Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
                  195 200 205
          Lys Ser Phe Asn Arg Gly Glu Cys
              210 215
           <![CDATA[ <210> 15]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 HCDR1]]>
           <![CDATA[ <400> 15]]>
          Asp Tyr Phe Leu His
          1 5
           <![CDATA[ <210> 16]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 HCDR2]]>
           <![CDATA[ <400> 16]]>
          Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Ala Gln Lys Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 17]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 HCDR3]]>
           <![CDATA[ <400> 17]]>
          Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr
          1 5 10
           <![CDATA[ <210> 18]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 VH]]>
           <![CDATA[ <400> 18]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
                      20 25 30
          Phe Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr
          65 70 75 80
          Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 19]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 LCDR1]]>
           <![CDATA[ <400> 19]]>
          Arg Ala Ser Gly Asn Ile Tyr Asn Tyr Leu Ala
          1 5 10
           <![CDATA[ <210> 20]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 LCDR2]]>
           <![CDATA[ <400> 20]]>
          Asp Ala Lys Thr Leu Ala Asp
          1 5
           <![CDATA[ <210> 21]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 LCDR3]]>
           <![CDATA[ <400> 21]]>
          Gln His Phe Trp Ser Leu Pro Phe Thr
          1 5
           <![CDATA[ <210> 22]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 VL]]>
           <![CDATA[ <400> 22]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Tyr Asn Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Val Ala Thr Tyr Tyr Cys Gln His Phe Trp Ser Leu Pro Phe
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
                      100 105
           <![CDATA[ <210> 23]]>
           <![CDATA[ <211> 674]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 VH-CH1(EE) - CD3 original/CD3 optimized VL-CH1 - Fc (Peg, PGLALA)]]>
           <![CDATA[ <400> 23]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
                      20 25 30
          Phe Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr
          65 70 75 80
          Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
                  115 120 125
          Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
              130 135 140
          Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val
          145 150 155 160
          Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
                          165 170 175
          Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
                      180 185 190
          Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
                  195 200 205
          Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
              210 215 220
          Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr
          225 230 235 240
          Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr
                          245 250 255
          Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp
                      260 265 270
          Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr
                  275 280 285
          Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu
              290 295 300
          Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu
          305 310 315 320
          Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly
                          325 330 335
          Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro
                      340 345 350
          Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
                  355 360 365
          Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
              370 375 380
          Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
          385 390 395 400
          Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
                          405 410 415
          Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
                      420 425 430
          His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
                  435 440 445
          Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala
              450 455 460
          Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
          465 470 475 480
          Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
                          485 490 495
          His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
                      500 505 510
          Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
                  515 520 525
          Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
              530 535 540
          Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro
          545 550 555 560
          Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
                          565 570 575
          Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val
                      580 585 590
          Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
                  595 600 605
          Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
              610 615 620
          Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
          625 630 635 640
          Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
                          645 650 655
          Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
                      660 665 670
          Ser Pro
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 449]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>TYRP1 VH-CH1(EE)-Fc (hole, PGLALA)]]>
           <![CDATA[ <400> 24]]>
          Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
          1 5 10 15
          Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Tyr
                      20 25 30
          Phe Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
                  35 40 45
          Gly Trp Ile Asn Pro Asp Asn Gly Asn Thr Val Tyr Ala Gln Lys Phe
              50 55 60
          Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Val Tyr
          65 70 75 80
          Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
                          85 90 95
          Thr Arg Arg Asp Tyr Thr Tyr Glu Lys Ala Ala Leu Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
                  115 120 125
          Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
              130 135 140
          Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val
          145 150 155 160
          Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
                          165 170 175
          Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
                      180 185 190
          Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
                  195 200 205
          Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys
              210 215 220
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
          225 230 235 240
          Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
                          245 250 255
          Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
                      260 265 270
          Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
                  275 280 285
          His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
              290 295 300
          Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
          305 310 315 320
          Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
                          325 330 335
          Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
                      340 345 350
          Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
                  355 360 365
          Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
              370 375 380
          Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
          385 390 395 400
          Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
                          405 410 415
          Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
                      420 425 430
          His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
                  435 440 445
          Pro
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 214]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TYRP1 VL-CL(RK)]]>
           <![CDATA[ <400> 25]]>
          Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
          1 5 10 15
          Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile Tyr Asn Tyr
                      20 25 30
          Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
                  35 40 45
          Tyr Asp Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly
              50 55 60
          Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
          65 70 75 80
          Glu Asp Val Ala Thr Tyr Tyr Cys Gln His Phe Trp Ser Leu Pro Phe
                          85 90 95
          Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
                      100 105 110
          Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys Ser Gly
                  115 120 125
          Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
              130 135 140
          Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
          145 150 155 160
          Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
                          165 170 175
          Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
                      180 185 190
          Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
                  195 200 205
          Phe Asn Arg Gly Glu Cys
              210
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 232]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CD3 Raw VH-CL]]>
           <![CDATA[ <400> 26]]>
          Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr
                      20 25 30
          Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
                      100 105 110
          Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val
                  115 120 125
          Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
              130 135 140
          Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
          145 150 155 160
          Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
                          165 170 175
          Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
                      180 185 190
          Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
                  195 200 205
          Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
              210 215 220
          Lys Ser Phe Asn Arg Gly Glu Cys
          225 230
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 232]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CD3 optimized VH-CL]]>
           <![CDATA[ <400> 27]]>
          Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr
                      20 25 30
          Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr
                      100 105 110
          Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val
                  115 120 125
          Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
              130 135 140
          Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
          145 150 155 160
          Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
                          165 170 175
          Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
                      180 185 190
          Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
                  195 200 205
          Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
              210 215 220
          Lys Ser Phe Asn Arg Gly Glu Cys
          225 230
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 360]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Human CD3 ε Stem - Fc(Knob) - Avi]]>
           <![CDATA[ <400> 28]]>
          Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys
          1 5 10 15
          Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro
                      20 25 30
          Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp
                  35 40 45
          Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys
              50 55 60
          Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg
          65 70 75 80
          Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg
                          85 90 95
          Val Ser Glu Asn Cys Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser
                      100 105 110
          Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
                  115 120 125
          Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
              130 135 140
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
          145 150 155 160
          Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                          165 170 175
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
                      180 185 190
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
                  195 200 205
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
              210 215 220
          Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
          225 230 235 240
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys
                          245 250 255
          Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
                      260 265 270
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
                  275 280 285
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
              290 295 300
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
          305 310 315 320
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                          325 330 335
          Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu
                      340 345 350
          Ala Gln Lys Ile Glu Trp His Glu
                  355 360
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 325]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Human CD3 delta Stem - Fc (hole) - Avi]]>
           <![CDATA[ <400> 29]]>
          Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys
          1 5 10 15
          Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser
                      20 25 30
          Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly
                  35 40 45
          Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr
              50 55 60
          Val Gln Val His Tyr Arg Met Cys Arg Ser Glu Gln Leu Tyr Phe Gln
          65 70 75 80
          Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
                          85 90 95
          Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
                      100 105 110
          Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
                  115 120 125
          His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
              130 135 140
          Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
          145 150 155 160
          Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
                          165 170 175
          Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
                      180 185 190
          Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
                  195 200 205
          Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
              210 215 220
          Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
          225 230 235 240
          Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
                          245 250 255
          Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr
                      260 265 270
          Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
                  275 280 285
          Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
              290 295 300
          Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys
          305 310 315 320
          Ile Glu Trp His Glu
                          325
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 351]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Cynomolgus CD3 ε Stem - Fc (Knob) - Avi]]>
           <![CDATA[ <400> 30]]>
          Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln
          1 5 10 15
          Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu
                      20 25 30
          Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser
                  35 40 45
          Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly
              50 55 60
          Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His
          65 70 75 80
          His Leu Tyr Leu Lys Ala Arg Val Ser Glu Asn Cys Val Asp Glu Gln
                          85 90 95
          Leu Tyr Phe Gln Gly Gly Ser Pro Lys Ser Ala Asp Lys Thr His Thr
                      100 105 110
          Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
                  115 120 125
          Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
              130 135 140
          Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
          145 150 155 160
          Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
                          165 170 175
          Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
                      180 185 190
          Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
                  195 200 205
          Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
              210 215 220
          Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
          225 230 235 240
          Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val
                          245 250 255
          Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
                      260 265 270
          Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
                  275 280 285
          Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
              290 295 300
          Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
          305 310 315 320
          Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly
                          325 330 335
          Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
                      340 345 350
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 334]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Cynomolgus monkey CD3 delta stem - Fc (hole) - Avi]]>
           <![CDATA[ <400> 31]]>
          Phe Lys Ile Pro Val Glu Glu Leu Glu Asp Arg Val Phe Val Lys Cys
          1 5 10 15
          Asn Thr Ser Val Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Thr
                      20 25 30
          Asn Asn Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly
                  35 40 45
          Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Ala
              50 55 60
          Val Gln Val His Tyr Arg Met Ser Gln Asn Cys Val Asp Glu Gln Leu
          65 70 75 80
          Tyr Phe Gln Gly Gly Ser Pro Lys Ser Ala Asp Lys Thr His Thr Cys
                          85 90 95
          Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
                      100 105 110
          Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
                  115 120 125
          Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
              130 135 140
          Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
          145 150 155 160
          Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
                          165 170 175
          Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
                      180 185 190
          Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
                  195 200 205
          Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser
              210 215 220
          Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys
          225 230 235 240
          Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
                          245 250 255
          Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
                      260 265 270
          Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
                  275 280 285
          Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
              290 295 300
          His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly
          305 310 315 320
          Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
                          325 330
           <![CDATA[ <210> 32]]>
           <![CDATA[ <211> 699]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Human TYRP1 ECD - Fc (Pestle) – Avi]]>
           <![CDATA[ <400> 32]]>
          Gln Phe Pro Arg Gln Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met
          1 5 10 15
          Cys Cys Pro Asp Leu Ser Pro Val Ser Gly Pro Gly Thr Asp Arg Cys
                      20 25 30
          Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser
                  35 40 45
          Arg Pro His Ser Pro Gln Tyr Pro His Asp Gly Arg Asp Asp Arg Glu
              50 55 60
          Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn
          65 70 75 80
          Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala
                          85 90 95
          Ala Cys Asp Gln Arg Val Leu Ile Val Arg Arg Asn Leu Leu Asp Leu
                      100 105 110
          Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys
                  115 120 125
          Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu
              130 135 140
          Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile
          145 150 155 160
          Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe
                          165 170 175
          Leu Gly Val Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu
                      180 185 190
          Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu
                  195 200 205
          Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr
              210 215 220
          Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp
          225 230 235 240
          Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn
                          245 250 255
          Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr
                      260 265 270
          Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Asp Gly Pro Ile Arg
                  275 280 285
          Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro
              290 295 300
          Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr
          305 310 315 320
          Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu
                          325 330 335
          Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu
                      340 345 350
          His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His
                  355 360 365
          Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp
              370 375 380
          Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr
          385 390 395 400
          Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met
                          405 410 415
          Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala
                      420 425 430
          Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Ile Gln Trp Pro Ser Arg Glu
                  435 440 445
          Phe Ser Val Pro Glu Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys
              450 455 460
          Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
          465 470 475 480
          Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
                          485 490 495
          Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
                      500 505 510
          Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
                  515 520 525
          Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
              530 535 540
          His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
          545 550 555 560
          Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
                          565 570 575
          Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu
                      580 585 590
          Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
                  595 600 605
          Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
              610 615 620
          Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
          625 630 635 640
          Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
                          645 650 655
          Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
                      660 665 670
          Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp
                  675 680 685
          Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
              690 695
           <![CDATA[ <210> 33]]>
           <![CDATA[ <211> 698]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Cynomolgus monkey TYRP1 ECD - Fc (pestle) – Avi]]>
           <![CDATA[ <400> 33]]>
          Gln Phe Pro Arg Glu Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met
          1 5 10 15
          Cys Cys Pro Asp Leu Ser Pro Met Ser Gly Pro Gly Thr Asp Arg Cys
                      20 25 30
          Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser
                  35 40 45
          Arg Pro His Ser Pro Arg Tyr Pro His Asp Gly Arg Asp Asp Arg Glu
              50 55 60
          Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn
          65 70 75 80
          Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala
                          85 90 95
          Ala Cys Asp Gln Arg Val Leu Val Val Arg Arg Asn Leu Leu Asp Leu
                      100 105 110
          Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys
                  115 120 125
          Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu
              130 135 140
          Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile
          145 150 155 160
          Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe
                          165 170 175
          Leu Gly Ala Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu
                      180 185 190
          Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu
                  195 200 205
          Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr
              210 215 220
          Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp
          225 230 235 240
          Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn
                          245 250 255
          Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr
                      260 265 270
          Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Ser Gly Pro Ile Arg
                  275 280 285
          Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro
              290 295 300
          Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr
          305 310 315 320
          Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu
                          325 330 335
          Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu
                      340 345 350
          His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His
                  355 360 365
          Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp
              370 375 380
          Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr
          385 390 395 400
          Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met
                          405 410 415
          Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala
                      420 425 430
          Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Val Gln Trp Pro Ser Arg Glu
                  435 440 445
          Phe Ser Val Pro Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
              450 455 460
          Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
          465 470 475 480
          Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
                          485 490 495
          Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
                      500 505 510
          Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
                  515 520 525
          Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
              530 535 540
          Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
          545 550 555 560
          Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
                          565 570 575
          Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Cys Arg Asp Glu Leu
                      580 585 590
          Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro
                  595 600 605
          Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
              610 615 620
          Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
          625 630 635 640
          Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
                          645 650 655
          Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
                      660 665 670
          Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile
                  675 680 685
          Phe Glu Ala Gln Lys Ile Glu Trp His Glu
              690 695
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 699]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Mouse TYRP1 ECD - Fc (Pestle) – Avi]]>
           <![CDATA[ <400> 34]]>
          Gln Phe Pro Arg Glu Cys Ala Asn Ile Glu Ala Leu Arg Arg Gly Val
          1 5 10 15
          Cys Cys Pro Asp Leu Leu Pro Ser Ser Gly Pro Gly Thr Asp Pro Cys
                      20 25 30
          Gly Ser Ser Ser Gly Arg Gly Arg Cys Val Ala Val Ile Ala Asp Ser
                  35 40 45
          Arg Pro His Ser Arg His Tyr Pro His Asp Gly Lys Asp Asp Arg Glu
              50 55 60
          Ala Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys Gln Cys Asn Asp Asn
          65 70 75 80
          Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala
                          85 90 95
          Ala Cys Asn Gln Lys Ile Leu Thr Val Arg Arg Asn Leu Leu Asp Leu
                      100 105 110
          Ser Pro Glu Glu Lys Ser His Phe Val Arg Ala Leu Asp Met Ala Lys
                  115 120 125
          Arg Thr Thr His Pro Gln Phe Val Ile Ala Thr Arg Arg Leu Glu Asp
              130 135 140
          Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Val
          145 150 155 160
          Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe
                          165 170 175
          Leu Gly Thr Gly Gln Glu Ser Phe Gly Asp Val Asp Phe Ser His Glu
                      180 185 190
          Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Gln Leu Glu
                  195 200 205
          Arg Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr
              210 215 220
          Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Val Cys Thr Asp Asp
          225 230 235 240
          Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn
                          245 250 255
          Ser Val Phe Ser Gln Trp Arg Val Val Cys Glu Ser Leu Glu Glu Tyr
                      260 265 270
          Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Gly Gly Pro Ile Arg
                  275 280 285
          Arg Asn Pro Ala Gly Asn Val Gly Arg Pro Ala Val Gln Arg Leu Pro
              290 295 300
          Glu Pro Gln Asp Val Thr Gln Cys Leu Glu Val Arg Val Phe Asp Thr
          305 310 315 320
          Pro Pro Phe Tyr Ser Asn Ser Thr Asp Ser Phe Arg Asn Thr Val Glu
                          325 330 335
          Gly Tyr Ser Ala Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu
                      340 345 350
          His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His
                  355 360 365
          Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp
              370 375 380
          Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr
          385 390 395 400
          Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met
                          405 410 415
          Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala
                      420 425 430
          Pro Asp Asn Leu Gly Tyr Ala Tyr Glu Val Gln Trp Pro Gly Gln Glu
                  435 440 445
          Phe Thr Val Ser Glu Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys
              450 455 460
          Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
          465 470 475 480
          Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
                          485 490 495
          Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
                      500 505 510
          Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
                  515 520 525
          Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
              530 535 540
          His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
          545 550 555 560
          Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
                          565 570 575
          Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu
                      580 585 590
          Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
                  595 600 605
          Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
              610 615 620
          Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
          625 630 635 640
          Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
                          645 650 655
          Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
                      660 665 670
          Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp
                  675 680 685
          Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
              690 695
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 225]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Fc (hole)]]>
           <![CDATA[ <400> 35]]>
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
          1 5 10 15
          Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
                      20 25 30
          Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
                  35 40 45
          Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
              50 55 60
          His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
          65 70 75 80
          Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
                          85 90 95
          Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
                      100 105 110
          Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
                  115 120 125
          Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
              130 135 140
          Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
          145 150 155 160
          Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
                          165 170 175
          Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
                      180 185 190
          Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
                  195 200 205
          His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser
              210 215 220
          Pro
          225
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 393]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII ECD - Avi - His]]>
           <![CDATA[ <400> 36]]>
          Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys
          1 5 10 15
          Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val
                      20 25 30
          Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly
                  35 40 45
          Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn
              50 55 60
          Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile
          65 70 75 80
          Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu
                          85 90 95
          Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly
                      100 105 110
          Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala
                  115 120 125
          Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln
              130 135 140
          Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg
          145 150 155 160
          Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys
                          165 170 175
          Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr
                      180 185 190
          Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys
                  195 200 205
          Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys
              210 215 220
          Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg
          225 230 235 240
          Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg
                          245 250 255
          Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu
                      260 265 270
          Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys
                  275 280 285
          Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys
              290 295 300
          Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala
          305 310 315 320
          Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly
                          325 330 335
          Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile
                      340 345 350
          Pro Ser Val Asp Gly Gly Ser Pro Thr Pro Pro Thr Pro Gly Gly Gly
                  355 360 365
          Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
              370 375 380
          Ala Arg Ala His His His His His His His
          385 390
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P056.021 HCDR1]]>
           <![CDATA[ <400> 37]]>
          Ser Tyr Trp Ile Ala
          1 5
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.021 HCDR2]]>
           <![CDATA[ <400> 38]]>
          Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.021 HCDR3]]>
           <![CDATA[ <400> 39]]>
          Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.021 VH]]>
           <![CDATA[ <400> 40]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 41]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.021 LCDR1]]>
           <![CDATA[ <400> 41]]>
          Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 42]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.021 LCDR2]]>
           <![CDATA[ <400> 42]]>
          Trp Ala Ser Thr Arg Glu Ser
          1 5
           <![CDATA[ <210> 43]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.021 LCDR3]]>
           <![CDATA[ <400> 43]]>
          Gln Gln Val His Ser Gly Pro Pro Val Thr
          1 5 10
           <![CDATA[ <210> 44]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.021 VL]]>
           <![CDATA[ <400> 44]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Val His Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu
                      100 105 110
          Ile Lys
           <![CDATA[ <210> 45]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P056.052 HCDR1]]>
           <![CDATA[ <400> 45]]>
          Asn Tyr Trp Ile Gly
          1 5
           <![CDATA[ <210> 46]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.052 HCDR2]]>
           <![CDATA[ <400> 46]]>
          Thr Ile Tyr Pro Gly Asp Ser Asp Arg Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 47]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.052 HCDR3]]>
           <![CDATA[ <400> 47]]>
          Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 48]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.052 VH]]>
           <![CDATA[ <400> 48]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Met Asn Tyr
                      20 25 30
          Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Thr Ile Tyr Pro Gly Asp Ser Asp Arg Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Leu Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 49]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.052 LCDR1]]>
           <![CDATA[ <400> 49]]>
          Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 50]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.052 LCDR2]]>
           <![CDATA[ <400> 50]]>
          Trp Ala Ser Thr Arg Glu Ser
          1 5
           <![CDATA[ <210> 51]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P056.052 LCDR3]]>
           <![CDATA[ <400> 51]]>
          Gln Gln Val His Ser Gly Pro Pro Val Thr
          1 5 10
           <![CDATA[ <210> 52]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.052 VL]]>
           <![CDATA[ <400> 52]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Val His Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu
                      100 105 110
          Ile Lys
           <![CDATA[ <210> 53]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P047.019 HCDR1]]>
           <![CDATA[ <400> 53]]>
          Ser Ile Trp Ile His
          1 5
           <![CDATA[ <210> 54]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P047.019 HCDR2]]>
           <![CDATA[ <400> 54]]>
          Thr Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 55]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P047.019 HCDR3]]>
           <![CDATA[ <400> 55]]>
          Thr Gly Pro Gly Leu Ala Phe Asp Tyr
          1 5
           <![CDATA[ <210> 56]]>
           <![CDATA[ <211> 118]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P047.019 VH]]>
           <![CDATA[ <400> 56]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Pro Ser Ile
                      20 25 30
          Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Thr Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Thr Gly Pro Gly Leu Ala Phe Asp Tyr Trp Gly Gln Gly Thr
                      100 105 110
          Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 57]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P047.019 LCDR1]]>
           <![CDATA[ <400> 57]]>
          Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 58]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P047.019 LCDR2]]>
           <![CDATA[ <400> 58]]>
          Trp Ala Ser Thr Arg Glu Ser
          1 5
           <![CDATA[ <210> 59]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P047.019 LCDR3]]>
           <![CDATA[ <400> 59]]>
          Gln Gln Ser Tyr Ser Thr Pro Ile Thr
          1 5
           <![CDATA[ <210> 60]]>
           <![CDATA[ <211> 113]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P047.019 VL]]>
           <![CDATA[ <400> 60]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Ser Tyr Ser Thr Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
                      100 105 110
          Lys
           <![CDATA[ <210> 61]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P057.012 HCDR1]]>
           <![CDATA[ <400> 61]]>
          Asn Tyr Trp Ile Ala
          1 5
           <![CDATA[ <210> 62]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.012 HCDR2]]>
           <![CDATA[ <400> 62]]>
          Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 63]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.012 HCDR3]]>
           <![CDATA[ <400> 63]]>
          Ala Thr Asn Ile Ala Ser Gly Gly Tyr Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 64]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.012 VH]]>
           <![CDATA[ <400> 64]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Ala Asn Tyr
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Ile Ile Tyr Pro Asp Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Thr Asn Ile Ala Ser Gly Gly Tyr Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 65]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P057.012 LCDR1]]>
           <![CDATA[ <400> 65]]>
          Lys Ser Ser Gln Ser Val Leu Trp Asn Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 66]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.012 LCDR2]]>
           <![CDATA[ <400> 66]]>
          Trp Ala Ser Lys Arg Glu Ser
          1 5
           <![CDATA[ <210> 67]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.012 LCDR3]]>
           <![CDATA[ <400> 67]]>
          Gln Gln Ser Tyr Ser Ala Pro Ile Thr
          1 5
           <![CDATA[ <210> 68]]>
           <![CDATA[ <211> 113]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P057.012 VL]]>
           <![CDATA[ <400> 68]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Trp Asn
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Lys Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Ser Tyr Ser Ala Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
                      100 105 110
          Lys
           <![CDATA[ <210> 69]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.011 HCDR1]]>
           <![CDATA[ <400> 69]]>
          Arg Arg Trp Ile Ala
          1 5
           <![CDATA[ <210> 70]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.011 HCDR2]]>
           <![CDATA[ <400> 70]]>
          Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 71]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.011 HCDR3]]>
           <![CDATA[ <400> 71]]>
          Ala Thr Asn Ile Ala Ser Gly Gly Tyr Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 72]]>
           <![CDATA[ <211> 121]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.011 VH]]>
           <![CDATA[ <400> 72]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Asn Phe Gly Arg Arg
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Ala Thr Asn Ile Ala Ser Gly Gly Tyr Phe Asp Tyr Trp Gly
                      100 105 110
          Gln Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 73]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P057.011 LCDR1]]>
           <![CDATA[ <400> 73]]>
          Lys Ser Ser Gln Ser Val Leu Trp Asn Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 74]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P057.011 LCDR2]]>
           <![CDATA[ <400> 74]]>
          Trp Ala Ser Lys Arg Glu Ser
          1 5
           <![CDATA[ <210> 75]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P057.011 LCDR3]]>
           <![CDATA[ <400> 75]]>
          Gln Gln Ser Tyr Ser Ala Pro Ile Thr
          1 5
           <![CDATA[ <210> 76]]>
           <![CDATA[ <211> 113]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P057.011 VL]]>
           <![CDATA[ <400> 76]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Trp Asn
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Lys Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Ser Tyr Ser Ala Pro Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
                      100 105 110
          Lys
           <![CDATA[ <210> 77]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.027 HCDR1]]>
           <![CDATA[ <400> 77]]>
          Asn Asn Trp Ile Ala
          1 5
           <![CDATA[ <210> 78]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.027 HCDR2]]>
           <![CDATA[ <400> 78]]>
          Val Ile Tyr Pro Gly Asp Ser Asp Lys Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 79]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.027 HCDR3]]>
           <![CDATA[ <400> 79]]>
          Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 80]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.027 VH]]>
           <![CDATA[ <400> 80]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Gly Asn Asn
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Val Ile Tyr Pro Gly Asp Ser Asp Lys Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 81]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.027 LCDR1]]>
           <![CDATA[ <400> 81]]>
          Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 82]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.027 LCDR2]]>
           <![CDATA[ <400> 82]]>
          Trp Ala Ser Thr Arg Glu Ser
          1 5
           <![CDATA[ <210> 83]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.027 LCDR3]]>
           <![CDATA[ <400> 83]]>
          Gln Gln Val His Ser Gly Pro Pro Val Thr
          1 5 10
           <![CDATA[ <210> 84]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P056.027 VL]]>
           <![CDATA[ <400> 84]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Val His Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu
                      100 105 110
          Ile Lys
           <![CDATA[ <210> 85]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P063.056 HCDR1]]>
           <![CDATA[ <400> 85]]>
          Ser Tyr Trp Ile Ala
          1 5
           <![CDATA[ <210> 86]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P063.056 HCDR2]]>
           <![CDATA[ <400> 86]]>
          Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 87]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P063.056 HCDR3]]>
           <![CDATA[ <400> 87]]>
          Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr
          1 5 10
           <![CDATA[ <210> 88]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P063.056 VH]]>
           <![CDATA[ <400> 88]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 89]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P063.056 LCDR1]]>
           <![CDATA[ <400> 89]]>
          Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 90]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P063.056 LCDR2]]>
           <![CDATA[ <400> 90]]>
          Trp Ala Ser Thr Arg Glu Ser
          1 5
           <![CDATA[ <210> 91]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P063.056 LCDR3]]>
           <![CDATA[ <400> 91]]>
          Gln Gln Gln Arg Asp Gly Pro Pro Val Thr
          1 5 10
           <![CDATA[ <210> 92]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P063.056 VL]]>
           <![CDATA[ <400> 92]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Gln Arg Asp Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu
                      100 105 110
          Ile Lys
           <![CDATA[ <210> 93]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P064.078 HCDR1]]>
           <![CDATA[ <400> 93]]>
          Ser Tyr Trp Ile Ala
          1 5
           <![CDATA[ <210> 94]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P064.078 HCDR2]]>
           <![CDATA[ <400> 94]]>
          Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 95]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P064.078 HCDR3]]>
           <![CDATA[ <400> 95]]>
          Val Ser Arg Leu Ser Tyr Ala Leu Asp Tyr
          1 5 10
           <![CDATA[ <210> 96]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P064.078 VH]]>
           <![CDATA[ <400> 96]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Ser Arg Leu Ser Tyr Ala Leu Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 97]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P064.078 LCDR1]]>
           <![CDATA[ <400> 97]]>
          Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 98]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P064.078 LCDR2]]>
           <![CDATA[ <400> 98]]>
          Trp Ala Ser Thr Arg Glu Ser
          1 5
           <![CDATA[ <210> 99]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P064.078 LCDR3]]>
           <![CDATA[ <400> 99]]>
          Gln Gln Val His Ser Gly Pro Pro Val Thr
          1 5 10
           <![CDATA[ <210> 100]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P064.078 VL]]>
           <![CDATA[ <400> 100]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Val His Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu
                      100 105 110
          Ile Lys
           <![CDATA[ <210> 101]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P065.036 HCDR1]]>
           <![CDATA[ <400> 101]]>
          Ser Tyr Trp Ile Ala
          1 5
           <![CDATA[ <210> 102]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P065.036 HCDR2]]>
           <![CDATA[ <400> 102]]>
          Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln
          1 5 10 15
          Gly
           <![CDATA[ <210> 103]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P065.036 HCDR3]]>
           <![CDATA[ <400> 103]]>
          Val Ser Arg Ser Ser Tyr Ala Leu Asp Tyr
          1 5 10
           <![CDATA[ <210> 104]]>
           <![CDATA[ <211> 119]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P065.036 VH]]>
           <![CDATA[ <400> 104]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Ser Arg Ser Ser Tyr Ala Leu Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser
                  115
           <![CDATA[ <210> 105]]>
           <![CDATA[ <211> 17]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P065.036 LCDR1]]>
           <![CDATA[ <400> 105]]>
          Lys Ser Ser Gln Ser Val Leu Tyr Ser Ser Asn Asn Lys Asn Tyr Leu
          1 5 10 15
          Ala
           <![CDATA[ <210> 106]]>
           <![CDATA[ <211> 7]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P065.036 LCDR2]]>
           <![CDATA[ <400> 106]]>
          Trp Ala Ser Thr Arg Glu Ser
          1 5
           <![CDATA[ <210> 107]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII P065.036 LCDR3]]>
           <![CDATA[ <400> 107]]>
          Gln Gln Val Tyr Ser Gly Pro Pro Val Thr
          1 5 10
           <![CDATA[ <210> 108]]>
           <![CDATA[ <211> 114]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>EGFRvIII P065.036 VL]]>
           <![CDATA[ <400> 108]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Val Tyr Ser Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu
                      100 105 110
          Ile Lys
           <![CDATA[ <210> 109]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII VH-CH1(EE) - CD3 original/CD3 optimized VL-CH1 - Fc (knob, PGLALA)]]>
           <![CDATA[ <400> 109]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
                  115 120 125
          Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
              130 135 140
          Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
          145 150 155 160
          Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
                          165 170 175
          Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
                      180 185 190
          Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
                  195 200 205
          Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Gly
              210 215 220
          Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu
          225 230 235 240
          Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu Thr Cys Gly
                          245 250 255
          Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn Trp Val Gln
                      260 265 270
          Glu Lys Pro Gly Gln Ala Phe Arg Gly Leu Ile Gly Gly Thr Asn Lys
                  275 280 285
          Arg Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly
              290 295 300
          Lys Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Glu
          305 310 315 320
          Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Leu Trp Val Phe Gly Gly Gly
                          325 330 335
          Thr Lys Leu Thr Val Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                      340 345 350
          Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
                  355 360 365
          Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
              370 375 380
          Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
          385 390 395 400
          Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
                          405 410 415
          Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
                      420 425 430
          Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
                  435 440 445
          Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
              450 455 460
          Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
          465 470 475 480
          Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
                          485 490 495
          Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
                      500 505 510
          Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
                  515 520 525
          Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
              530 535 540
          Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
          545 550 555 560
          Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
                          565 570 575
          Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
                      580 585 590
          Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
                  595 600 605
          Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
              610 615 620
          Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
          625 630 635 640
          Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
                          645 650 655
          Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                      660 665 670
           <![CDATA[ <210> 110]]>
           <![CDATA[ <211> 447]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII VH-CH1(EE)-Fc (hole, PGLALA)]]>
           <![CDATA[ <400> 110]]>
          Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
          1 5 10 15
          Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Asp Ser Tyr
                      20 25 30
          Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
                  35 40 45
          Gly Val Ile His Pro Tyr Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe
              50 55 60
          Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
          65 70 75 80
          Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
                          85 90 95
          Ala Arg Val Ser Arg Ser Ser Tyr Ala Phe Asp Tyr Trp Gly Gln Gly
                      100 105 110
          Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
                  115 120 125
          Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
              130 135 140
          Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
          145 150 155 160
          Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
                          165 170 175
          Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
                      180 185 190
          Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
                  195 200 205
          Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys
              210 215 220
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
          225 230 235 240
          Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
                          245 250 255
          Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
                      260 265 270
          Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
                  275 280 285
          Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
              290 295 300
          Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
          305 310 315 320
          Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
                          325 330 335
          Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr
                      340 345 350
          Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser
                  355 360 365
          Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
              370 375 380
          Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
          385 390 395 400
          Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys
                          405 410 415
          Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
                      420 425 430
          Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  435 440 445
           <![CDATA[ <210> 111]]>
           <![CDATA[ <211> 221]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> EGFRvIII VL-CL(RK)]]>
           <![CDATA[ <400> 111]]>
          Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
          1 5 10 15
          Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser
                      20 25 30
          Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
                  35 40 45
          Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
              50 55 60
          Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
          65 70 75 80
          Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
                          85 90 95
          Gln Arg Asp Gly Pro Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu
                      100 105 110
          Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
                  115 120 125
          Asp Arg Lys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
              130 135 140
          Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
          145 150 155 160
          Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
                          165 170 175
          Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
                      180 185 190
          Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
                  195 200 205
          Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
              210 215 220
           <![CDATA[ <210> 112]]>
           <![CDATA[ <211> 186]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 112]]>
          Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys
          1 5 10 15
          Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro
                      20 25 30
          Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp
                  35 40 45
          Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys
              50 55 60
          Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg
          65 70 75 80
          Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg
                          85 90 95
          Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile
                      100 105 110
          Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr
                  115 120 125
          Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly
              130 135 140
          Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro
          145 150 155 160
          Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp
                          165 170 175
          Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile
                      180 185
           <![CDATA[ <210> 113]]>
           <![CDATA[ <211> 177]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Crab-eating Macaque]]>
           <![CDATA[ <400> 113]]>
          Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln
          1 5 10 15
          Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu
                      20 25 30
          Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser
                  35 40 45
          Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly
              50 55 60
          Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His
          65 70 75 80
          His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp
                          85 90 95
          Val Met Ala Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Leu
                      100 105 110
          Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys
                  115 120 125
          Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly
              130 135 140
          Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro
          145 150 155 160
          Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg
                          165 170 175
          Ile
           <![CDATA[ <210> 114]]>
           <![CDATA[ <211> 513]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 114]]>
          Gln Phe Pro Arg Gln Cys Ala Thr Val Glu Ala Leu Arg Ser Gly Met
          1 5 10 15
          Cys Cys Pro Asp Leu Ser Pro Val Ser Gly Pro Gly Thr Asp Arg Cys
                      20 25 30
          Gly Ser Ser Ser Gly Arg Gly Arg Cys Glu Ala Val Thr Ala Asp Ser
                  35 40 45
          Arg Pro His Ser Pro Gln Tyr Pro His Asp Gly Arg Asp Asp Arg Glu
              50 55 60
          Val Trp Pro Leu Arg Phe Phe Asn Arg Thr Cys His Cys Asn Gly Asn
          65 70 75 80
          Phe Ser Gly His Asn Cys Gly Thr Cys Arg Pro Gly Trp Arg Gly Ala
                          85 90 95
          Ala Cys Asp Gln Arg Val Leu Ile Val Arg Arg Asn Leu Leu Asp Leu
                      100 105 110
          Ser Lys Glu Glu Lys Asn His Phe Val Arg Ala Leu Asp Met Ala Lys
                  115 120 125
          Arg Thr Thr His Pro Leu Phe Val Ile Ala Thr Arg Arg Ser Glu Glu
              130 135 140
          Ile Leu Gly Pro Asp Gly Asn Thr Pro Gln Phe Glu Asn Ile Ser Ile
          145 150 155 160
          Tyr Asn Tyr Phe Val Trp Thr His Tyr Tyr Ser Val Lys Lys Thr Phe
                          165 170 175
          Leu Gly Val Gly Gln Glu Ser Phe Gly Glu Val Asp Phe Ser His Glu
                      180 185 190
          Gly Pro Ala Phe Leu Thr Trp His Arg Tyr His Leu Leu Arg Leu Glu
                  195 200 205
          Lys Asp Met Gln Glu Met Leu Gln Glu Pro Ser Phe Ser Leu Pro Tyr
              210 215 220
          Trp Asn Phe Ala Thr Gly Lys Asn Val Cys Asp Ile Cys Thr Asp Asp
          225 230 235 240
          Leu Met Gly Ser Arg Ser Asn Phe Asp Ser Thr Leu Ile Ser Pro Asn
                          245 250 255
          Ser Val Phe Ser Gln Trp Arg Val Val Cys Asp Ser Leu Glu Asp Tyr
                      260 265 270
          Asp Thr Leu Gly Thr Leu Cys Asn Ser Thr Glu Asp Gly Pro Ile Arg
                  275 280 285
          Arg Asn Pro Ala Gly Asn Val Ala Arg Pro Met Val Gln Arg Leu Pro
              290 295 300
          Glu Pro Gln Asp Val Ala Gln Cys Leu Glu Val Gly Leu Phe Asp Thr
          305 310 315 320
          Pro Pro Phe Tyr Ser Asn Ser Thr Asn Ser Phe Arg Asn Thr Val Glu
                          325 330 335
          Gly Tyr Ser Asp Pro Thr Gly Lys Tyr Asp Pro Ala Val Arg Ser Leu
                      340 345 350
          His Asn Leu Ala His Leu Phe Leu Asn Gly Thr Gly Gly Gln Thr His
                  355 360 365
          Leu Ser Pro Asn Asp Pro Ile Phe Val Leu Leu His Thr Phe Thr Asp
              370 375 380
          Ala Val Phe Asp Glu Trp Leu Arg Arg Tyr Asn Ala Asp Ile Ser Thr
          385 390 395 400
          Phe Pro Leu Glu Asn Ala Pro Ile Gly His Asn Arg Gln Tyr Asn Met
                          405 410 415
          Val Pro Phe Trp Pro Pro Val Thr Asn Thr Glu Met Phe Val Thr Ala
                      420 425 430
          Pro Asp Asn Leu Gly Tyr Thr Tyr Glu Ile Gln Trp Pro Ser Arg Glu
                  435 440 445
          Phe Ser Val Pro Glu Ile Ile Ala Ile Ala Val Val Gly Ala Leu Leu
              450 455 460
          Leu Val Ala Leu Ile Phe Gly Thr Ala Ser Tyr Leu Ile Arg Ala Arg
          465 470 475 480
          Arg Ser Met Asp Glu Ala Asn Gln Pro Leu Leu Thr Asp Gln Tyr Gln
                          485 490 495
          Cys Tyr Ala Glu Glu Tyr Glu Lys Leu Gln Asn Pro Asn Gln Ser Val
                      500 505 510
          Val
           <![CDATA[ <210> 115]]>
           <![CDATA[ <211> 919]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 115]]>
          Leu Glu Glu Lys Lys Gly Asn Tyr Val Val Thr Asp His Gly Ser Cys
          1 5 10 15
          Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val
                      20 25 30
          Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly
                  35 40 45
          Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn
              50 55 60
          Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile
          65 70 75 80
          Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu
                          85 90 95
          Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly
                      100 105 110
          Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala
                  115 120 125
          Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln
              130 135 140
          Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg
          145 150 155 160
          Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys
                          165 170 175
          Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr
                      180 185 190
          Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys
                  195 200 205
          Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys
              210 215 220
          Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg
          225 230 235 240
          Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg
                          245 250 255
          Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu
                      260 265 270
          Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys
                  275 280 285
          Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys
              290 295 300
          Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala
          305 310 315 320
          Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly
                          325 330 335
          Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile
                      340 345 350
          Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val
                  355 360 365
          Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His Ile Val Arg
              370 375 380
          Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu Val Glu Pro
          385 390 395 400
          Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu
                          405 410 415
          Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser Gly Ala Phe
                      420 425 430
          Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys
                  435 440 445
          Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser Pro Lys Ala
              450 455 460
          Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser Val Asp Asn
          465 470 475 480
          Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln
                          485 490 495
          Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg
                      500 505 510
          Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn Trp Cys Val
                  515 520 525
          Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg Leu Val His
              530 535 540
          Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln His Val
          545 550 555 560
          Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys
                          565 570 575
          Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu
                      580 585 590
          Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp Val Trp Ser
                  595 600 605
          Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser Lys Pro Tyr
              610 615 620
          Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu Lys Gly Glu
          625 630 635 640
          Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met
                          645 650 655
          Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe Arg Glu
                      660 665 670
          Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln Arg Tyr Leu
                  675 680 685
          Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro Thr Asp Ser
              690 695 700
          Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp Asp Val Val
          705 710 715 720
          Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe Phe Ser Ser Pro
                          725 730 735
          Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu Ser Ala Thr Ser Asn
                      740 745 750
          Asn Ser Thr Val Ala Cys Ile Asp Arg Asn Gly Leu Gln Ser Cys Pro
                  755 760 765
          Ile Lys Glu Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp Pro Thr Gly
              770 775 780
          Ala Leu Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro Val Pro Glu
          785 790 795 800
          Tyr Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser Val Gln Asn
                          805 810 815
          Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro
                      820 825 830
          His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu
                  835 840 845
          Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala
              850 855 860
          His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp
          865 870 875 880
          Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe
                          885 890 895
          Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln
                      900 905 910
          Ser Ser Glu Phe Ile Gly Ala
                  915
           <![CDATA[ <210> 116]]>
           <![CDATA[ <211> 1186]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 116]]>
          Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr Gln
          1 5 10 15
          Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe Asn
                      20 25 30
          Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln Arg
                  35 40 45
          Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly Tyr
              50 55 60
          Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn Leu
          65 70 75 80
          Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu Ala
                          85 90 95
          Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu Pro
                      100 105 110
          Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser Asn
                  115 120 125
          Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile Val
              130 135 140
          Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His Leu
          145 150 155 160
          Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys Trp
                          165 170 175
          Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys Ala
                      180 185 190
          Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys Cys
                  195 200 205
          His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp Cys
              210 215 220
          Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr Cys
          225 230 235 240
          Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val Asn
                          245 250 255
          Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys Pro
                      260 265 270
          Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys Gly
                  275 280 285
          Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys Lys
              290 295 300
          Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu
          305 310 315 320
          Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys
                          325 330 335
          Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe
                      340 345 350
          Arg Gly Asp Ser Phe Thr His Thr Pro Leu Asp Pro Gln Glu Leu
                  355 360 365
          Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln
              370 375 380
          Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu
          385 390 395 400
          Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val
                          405 410 415
          Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile
                      420 425 430
          Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala
                  435 440 445
          Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr
              450 455 460
          Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln
          465 470 475 480
          Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro
                          485 490 495
          Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val
                      500 505 510
          Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn
                  515 520 525
          Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn
              530 535 540
          Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His
          545 550 555 560
          Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met
                          565 570 575
          Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val
                      580 585 590
          Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly
                  595 600 605
          Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr
              610 615 620
          Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile
          625 630 635 640
          Gly Leu Phe Met Arg Arg Arg His Ile Val Arg Lys Arg Thr Leu Arg
                          645 650 655
          Arg Leu Leu Gln Glu Arg Glu Leu Val Glu Pro Leu Thr Pro Ser Gly
                      660 665 670
          Glu Ala Pro Asn Gln Ala Leu Leu Arg Ile Leu Lys Glu Thr Glu Phe
                  675 680 685
          Lys Lys Ile Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys
              690 695 700
          Gly Leu Trp Ile Pro Glu Gly Glu Lys Val Lys Ile Pro Val Ala Ile
          705 710 715 720
          Lys Glu Leu Arg Glu Ala Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu
                          725 730 735
          Asp Glu Ala Tyr Val Met Ala Ser Val Asp Asn Pro His Val Cys Arg
                      740 745 750
          Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Ile Thr Gln Leu
                  755 760 765
          Met Pro Phe Gly Cys Leu Leu Asp Tyr Val Arg Glu His Lys Asp Asn
              770 775 780
          Ile Gly Ser Gln Tyr Leu Leu Asn Trp Cys Val Gln Ile Ala Lys Gly
          785 790 795 800
          Met Asn Tyr Leu Glu Asp Arg Arg Leu Val His Arg Asp Leu Ala Ala
                          805 810 815
          Arg Asn Val Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe
                      820 825 830
          Gly Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu
                  835 840 845
          Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His
              850 855 860
          Arg Ile Tyr Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val
          865 870 875 880
          Trp Glu Leu Met Thr Phe Gly Ser Lys Pro Tyr Asp Gly Ile Pro Ala
                          885 890 895
          Ser Glu Ile Ser Ser Ile Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro
                      900 905 910
          Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met
                  915 920 925
          Ile Asp Ala Asp Ser Arg Pro Lys Phe Arg Glu Leu Ile Ile Glu Phe
              930 935 940
          Ser Lys Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile Gln Gly Asp
          945 950 955 960
          Glu Arg Met His Leu Pro Ser Pro Thr Asp Ser Asn Phe Tyr Arg Ala
                          965 970 975
          Leu Met Asp Glu Glu Asp Met Asp Asp Val Val Asp Ala Asp Glu Tyr
                      980 985 990
          Leu Ile Pro Gln Gln Gly Phe Phe Ser Ser Pro Ser Thr Ser Arg Thr
                  995 1000 1005
          Pro Leu Leu Ser Ser Leu Ser Ala Thr Ser Asn Asn Ser Thr Val
              1010 1015 1020
          Ala Cys Ile Asp Arg Asn Gly Leu Gln Ser Cys Pro Ile Lys Glu
              1025 1030 1035
          Asp Ser Phe Leu Gln Arg Tyr Ser Ser Asp Pro Thr Gly Ala Leu
              1040 1045 1050
          Thr Glu Asp Ser Ile Asp Asp Thr Phe Leu Pro Val Pro Glu Tyr
              1055 1060 1065
          Ile Asn Gln Ser Val Pro Lys Arg Pro Ala Gly Ser Val Gln Asn
              1070 1075 1080
          Pro Val Tyr His Asn Gln Pro Leu Asn Pro Ala Pro Ser Arg Asp
              1085 1090 1095
          Pro His Tyr Gln Asp Pro His Ser Thr Ala Val Gly Asn Pro Glu
              1100 1105 1110
          Tyr Leu Asn Thr Val Gln Pro Thr Cys Val Asn Ser Thr Phe Asp
              1115 1120 1125
          Ser Pro Ala His Trp Ala Gln Lys Gly Ser His Gln Ile Ser Leu
              1130 1135 1140
          Asp Asn Pro Asp Tyr Gln Gln Asp Phe Phe Pro Lys Glu Ala Lys
              1145 1150 1155
          Pro Asn Gly Ile Phe Lys Gly Ser Thr Ala Glu Asn Ala Glu Tyr
              1160 1165 1170
          Leu Arg Val Ala Pro Gln Ser Ser Glu Phe Ile Gly Ala
              1175 1180 1185
           <![CDATA[ <210> 117]]>
           <![CDATA[ <211> 225]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> hIgG1 Fc region]]>
           <![CDATA[ <400> 117]]>
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
          1 5 10 15
          Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
                      20 25 30
          Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
                  35 40 45
          Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
              50 55 60
          His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
          65 70 75 80
          Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
                          85 90 95
          Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
                      100 105 110
          Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
                  115 120 125
          Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
              130 135 140
          Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
          145 150 155 160
          Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
                          165 170 175
          Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
                      180 185 190
          Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
                  195 200 205
          His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
              210 215 220
          Pro
          225
           <![CDATA[ <210> 118]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker GGGGSGGGGS]]>
           <![CDATA[ <400> 118]]>
          Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
          1 5 10
           <![CDATA[ <210> 119]]>
           <![CDATA[ <211> 11]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker DGGGGSGGGGS]]>
           <![CDATA[ <400> 119]]>
          Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
          1 5 10
           <![CDATA[ <210> 120]]>
           <![CDATA[ <211> 107]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Human kappa CL domain]]>
           <![CDATA[ <400> 120]]>
          Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
          1 5 10 15
          Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
                      20 25 30
          Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
                  35 40 45
          Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
              50 55 60
          Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
          65 70 75 80
          Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
                          85 90 95
          Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
                      100 105
           <![CDATA[ <210> 121]]>
           <![CDATA[ <211> 105]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Human λ CL domain]]>
           <![CDATA[ <400> 121]]>
          Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu
          1 5 10 15
          Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe
                      20 25 30
          Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val
                  35 40 45
          Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys
              50 55 60
          Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser
          65 70 75 80
          His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu
                          85 90 95
          Lys Thr Val Ala Pro Thr Glu Cys Ser
                      100 105
           <![CDATA[ <210> 122]]>
           <![CDATA[ <211> 328]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Human IgG1 heavy chain constant region (CH1-CH2-CH3)]]>
           <![CDATA[ <400> 122]]>
          Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
          1 5 10 15
          Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
                      20 25 30
          Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
                  35 40 45
          Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
              50 55 60
          Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
          65 70 75 80
          Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
                          85 90 95
          Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
                      100 105 110
          Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
                  115 120 125
          Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
              130 135 140
          Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
          145 150 155 160
          Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
                          165 170 175
          Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
                      180 185 190
          His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
                  195 200 205
          Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
              210 215 220
          Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
          225 230 235 240
          Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
                          245 250 255
          Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
                      260 265 270
          Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
                  275 280 285
          Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
              290 295 300
          Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
          305 310 315 320
          Gln Lys Ser Leu Ser Leu Ser Pro
                          325
           <![CDATA[ <210> 123]]>
           <![CDATA[ <211> 120]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> 16D5 VH]]>
           <![CDATA[ <400> 123]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser
                  115 120
           <![CDATA[ <210> 124]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> 16D5 CDRH1]]>
           <![CDATA[ <400> 124]]>
          Asn Ala Trp Met Ser
          1 5
           <![CDATA[ <210> 125]]>
           <![CDATA[ <211> 19]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> 16D5 CDRH2]]>
           <![CDATA[ <400> 125]]>
          Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro
          1 5 10 15
          Val Lys Gly
           <![CDATA[ <210> 126]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> 16D5 CDRH3]]>
           <![CDATA[ <400> 126]]>
          Pro Trp Glu Trp Ser Trp Tyr Asp Tyr
          1 5
           <![CDATA[ <210> 127]]>
           <![CDATA[ <211> 687]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 optimized 2+1 canonical form K chain]]>
           <![CDATA[ <400> 127]]>
          Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr
                      20 25 30
          Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr
                      100 105 110
          Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
                  115 120 125
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
              130 135 140
          Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
          145 150 155 160
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
                          165 170 175
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
                      180 185 190
          Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
                  195 200 205
          Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
              210 215 220
          Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu
          225 230 235 240
          Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser
                          245 250 255
          Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp
                      260 265 270
          Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly
                  275 280 285
          Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro
              290 295 300
          Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu
          305 310 315 320
          Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr
                          325 330 335
          Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln Gly
                      340 345 350
          Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
                  355 360 365
          Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
              370 375 380
          Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
          385 390 395 400
          Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
                          405 410 415
          Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
                      420 425 430
          Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
                  435 440 445
          Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
              450 455 460
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
          465 470 475 480
          Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
                          485 490 495
          Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
                      500 505 510
          Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
                  515 520 525
          Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
              530 535 540
          Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
          545 550 555 560
          Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
                          565 570 575
          Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
                      580 585 590
          Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
                  595 600 605
          Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
              610 615 620
          Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
          625 630 635 640
          Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
                          645 650 655
          Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
                      660 665 670
          Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  675 680 685
           <![CDATA[ <210> 128]]>
           <![CDATA[ <211> 448]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 optimized 2+1 canonical form H chain]]>
           <![CDATA[ <400> 128]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                  115 120 125
          Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
              130 135 140
          Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
          145 150 155 160
          Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
                          165 170 175
          Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
                      180 185 190
          Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
                  195 200 205
          Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
              210 215 220
          Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
          225 230 235 240
          Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
                          245 250 255
          Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
                      260 265 270
          Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
                  275 280 285
          Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
              290 295 300
          Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
          305 310 315 320
          Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
                          325 330 335
          Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
                      340 345 350
          Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
                  355 360 365
          Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
              370 375 380
          Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
          385 390 395 400
          Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp
                          405 410 415
          Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
                      420 425 430
          Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  435 440 445
           <![CDATA[ <210> 129]]>
           <![CDATA[ <211> 215]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Common light chains]]>
           <![CDATA[ <400> 129]]>
          Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly
          1 5 10 15
          Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser
                      20 25 30
          Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Phe Arg Gly
                  35 40 45
          Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Thr Pro Ala Arg Phe
              50 55 60
          Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala
          65 70 75 80
          Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn
                          85 90 95
          Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
                      100 105 110
          Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu
                  115 120 125
          Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro
              130 135 140
          Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala
          145 150 155 160
          Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala
                          165 170 175
          Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg
                      180 185 190
          Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr
                  195 200 205
          Val Ala Pro Thr Glu Cys Ser
              210 215
           <![CDATA[ <210> 130]]>
           <![CDATA[ <211> 687]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 Optimized 2+1 Inversion K-chain]]>
           <![CDATA[ <400> 130]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                  115 120 125
          Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
              130 135 140
          Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
          145 150 155 160
          Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
                          165 170 175
          Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
                      180 185 190
          Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
                  195 200 205
          Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
              210 215 220
          Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu
          225 230 235 240
          Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
                          245 250 255
          Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr Ala Met Asn Trp Val Arg
                      260 265 270
          Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys
                  275 280 285
          Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
              290 295 300
          Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
          305 310 315 320
          Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Thr
                          325 330 335
          Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr Gly Tyr Trp Gly Gln Gly
                      340 345 350
          Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
                  355 360 365
          Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
              370 375 380
          Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
          385 390 395 400
          Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
                          405 410 415
          Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
                      420 425 430
          Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
                  435 440 445
          Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
              450 455 460
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
          465 470 475 480
          Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
                          485 490 495
          Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
                      500 505 510
          Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
                  515 520 525
          Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
              530 535 540
          Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
          545 550 555 560
          Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
                          565 570 575
          Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
                      580 585 590
          Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
                  595 600 605
          Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
              610 615 620
          Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
          625 630 635 640
          Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
                          645 650 655
          Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
                      660 665 670
          Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  675 680 685
           <![CDATA[ <210> 131]]>
           <![CDATA[ <211> 687]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 Optimised 1+1 head to tail inverted form K chain]]>
           <![CDATA[ <400> 131]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                  115 120 125
          Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
              130 135 140
          Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
          145 150 155 160
          Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
                          165 170 175
          Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
                      180 185 190
          Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
                  195 200 205
          Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
              210 215 220
          Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu
          225 230 235 240
          Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
                          245 250 255
          Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr Ala Met Asn Trp Val Arg
                      260 265 270
          Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Arg Ile Arg Ser Lys
                  275 280 285
          Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe
              290 295 300
          Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn
          305 310 315 320
          Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val Arg His Thr
                          325 330 335
          Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr Gly Tyr Trp Gly Gln Gly
                      340 345 350
          Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
                  355 360 365
          Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
              370 375 380
          Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
          385 390 395 400
          Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
                          405 410 415
          Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
                      420 425 430
          Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
                  435 440 445
          Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
              450 455 460
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
          465 470 475 480
          Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
                          485 490 495
          Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
                      500 505 510
          Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
                  515 520 525
          Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
              530 535 540
          Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
          545 550 555 560
          Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
                          565 570 575
          Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
                      580 585 590
          Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
                  595 600 605
          Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
              610 615 620
          Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
          625 630 635 640
          Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
                          645 650 655
          Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
                      660 665 670
          Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  675 680 685
           <![CDATA[ <210> 132]]>
           <![CDATA[ <211> 225]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 optimized 1+1 head to tail inverted form H chain]]>
           <![CDATA[ <400> 132]]>
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
          1 5 10 15
          Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
                      20 25 30
          Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
                  35 40 45
          Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
              50 55 60
          His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
          65 70 75 80
          Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
                          85 90 95
          Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
                      100 105 110
          Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
                  115 120 125
          Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
              130 135 140
          Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
          145 150 155 160
          Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
                          165 170 175
          Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
                      180 185 190
          Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
                  195 200 205
          His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
              210 215 220
          Pro
          225
           <![CDATA[ <210> 133]]>
           <![CDATA[ <211> 687]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 optimized 1+1 head to tail canonical form K chain]]>
           <![CDATA[ <400> 133]]>
          Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gln Phe Ser Ser Tyr
                      20 25 30
          Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp
              50 55 60
          Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Val Arg His Thr Thr Phe Pro Ser Ser Tyr Val Ser Tyr Tyr
                      100 105 110
          Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
                  115 120 125
          Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
              130 135 140
          Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
          145 150 155 160
          Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
                          165 170 175
          Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
                      180 185 190
          Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
                  195 200 205
          Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
              210 215 220
          Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Glu
          225 230 235 240
          Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser
                          245 250 255
          Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala Trp
                      260 265 270
          Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly
                  275 280 285
          Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala Pro
              290 295 300
          Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu
          305 310 315 320
          Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr
                          325 330 335
          Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln Gly
                      340 345 350
          Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
                  355 360 365
          Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
              370 375 380
          Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
          385 390 395 400
          Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
                          405 410 415
          Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
                      420 425 430
          Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro
                  435 440 445
          Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
              450 455 460
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
          465 470 475 480
          Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
                          485 490 495
          Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
                      500 505 510
          Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
                  515 520 525
          Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
              530 535 540
          Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
          545 550 555 560
          Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys
                          565 570 575
          Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
                      580 585 590
          Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp
                  595 600 605
          Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
              610 615 620
          Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
          625 630 635 640
          Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
                          645 650 655
          Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
                      660 665 670
          Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  675 680 685
           <![CDATA[ <210> 134]]>
           <![CDATA[ <211> 225]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 optimized 1+1 head to tail canonical form H chain]]>
           <![CDATA[ <400> 134]]>
          Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly
          1 5 10 15
          Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
                      20 25 30
          Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
                  35 40 45
          Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
              50 55 60
          His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
          65 70 75 80
          Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
                          85 90 95
          Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile
                      100 105 110
          Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
                  115 120 125
          Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
              130 135 140
          Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
          145 150 155 160
          Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
                          165 170 175
          Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
                      180 185 190
          Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
                  195 200 205
          His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
              210 215 220
          Pro
          225
           <![CDATA[ <210> 135]]>
           <![CDATA[ <211> 448]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 optimized 1+1 IgG-like format K chain]]>
           <![CDATA[ <400> 135]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                  115 120 125
          Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
              130 135 140
          Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
          145 150 155 160
          Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
                          165 170 175
          Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
                      180 185 190
          Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
                  195 200 205
          Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
              210 215 220
          Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
          225 230 235 240
          Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
                          245 250 255
          Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
                      260 265 270
          Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
                  275 280 285
          Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
              290 295 300
          Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
          305 310 315 320
          Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
                          325 330 335
          Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
                      340 345 350
          Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
                  355 360 365
          Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
              370 375 380
          Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
          385 390 395 400
          Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
                          405 410 415
          Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
                      420 425 430
          Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  435 440 445
           <![CDATA[ <210> 136]]>
           <![CDATA[ <211> 448]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial sequences]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> FOLR1 TCB 16D5-CD3 optimized 1+1 IgG-like format H chain]]>
           <![CDATA[ <400> 136]]>
          Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
          1 5 10 15
          Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala
                      20 25 30
          Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
                  35 40 45
          Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala
              50 55 60
          Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr
          65 70 75 80
          Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
                          85 90 95
          Tyr Cys Thr Thr Pro Trp Glu Trp Ser Trp Tyr Asp Tyr Trp Gly Gln
                      100 105 110
          Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
                  115 120 125
          Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
              130 135 140
          Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
          145 150 155 160
          Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
                          165 170 175
          Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
                      180 185 190
          Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
                  195 200 205
          Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
              210 215 220
          Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
          225 230 235 240
          Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
                          245 250 255
          Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
                      260 265 270
          Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
                  275 280 285
          Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
              290 295 300
          Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
          305 310 315 320
          Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu
                          325 330 335
          Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
                      340 345 350
          Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
                  355 360 365
          Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
              370 375 380
          Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
          385 390 395 400
          Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp
                          405 410 415
          Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
                      420 425 430
          Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  435 440 445
           <![CDATA[ <210> 137]]>
           <![CDATA[ <211> 257]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Homo sapiens]]>
           <![CDATA[ <400> 137]]>
          Met Ala Gln Arg Met Thr Thr Gln Leu Leu Leu Leu Leu Val Trp Val
          1 5 10 15
          Ala Val Val Gly Glu Ala Gln Thr Arg Ile Ala Trp Ala Arg Thr Glu
                      20 25 30
          Leu Leu Asn Val Cys Met Asn Ala Lys His His Lys Glu Lys Pro Gly
                  35 40 45
          Pro Glu Asp Lys Leu His Glu Gln Cys Arg Pro Trp Arg Lys Asn Ala
              50 55 60
          Cys Cys Ser Thr Asn Thr Ser Gln Glu Ala His Lys Asp Val Ser Tyr
          65 70 75 80
          Leu Tyr Arg Phe Asn Trp Asn His Cys Gly Glu Met Ala Pro Ala Cys
                          85 90 95
          Lys Arg His Phe Ile Gln Asp Thr Cys Leu Tyr Glu Cys Ser Pro Asn
                      100 105 110
          Leu Gly Pro Trp Ile Gln Gln Val Asp Gln Ser Trp Arg Lys Glu Arg
                  115 120 125
          Val Leu Asn Val Pro Leu Cys Lys Glu Asp Cys Glu Gln Trp Trp Glu
              130 135 140
          Asp Cys Arg Thr Ser Tyr Thr Cys Lys Ser Asn Trp His Lys Gly Trp
          145 150 155 160
          Asn Trp Thr Ser Gly Phe Asn Lys Cys Ala Val Gly Ala Ala Cys Gln
                          165 170 175
          Pro Phe His Phe Tyr Phe Pro Thr Pro Thr Val Leu Cys Asn Glu Ile
                      180 185 190
          Trp Thr His Ser Tyr Lys Val Ser Asn Tyr Ser Arg Gly Ser Gly Arg
                  195 200 205
          Cys Ile Gln Met Trp Phe Asp Pro Ala Gln Gly Asn Pro Asn Glu Glu
              210 215 220
          Val Ala Arg Phe Tyr Ala Ala Ala Met Ser Gly Ala Gly Pro Trp Ala
          225 230 235 240
          Ala Trp Pro Phe Leu Leu Ser Leu Ala Leu Met Leu Leu Trp Leu Leu
                          245 250 255
          Ser
          
      

Claims (31)

A bispecific antibody that binds CD3 and folate receptor 1 (FolR1), wherein the bispecific antibody comprises (i) a first antigen-binding domain capable of specifically binding to CD3, the first antigen-binding domain comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 2 , HCDR 2 of SEQ ID NO: 3 and HCDR 3 of SEQ ID NO: 5; and a light chain variable region (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 8, SEQ ID NO : LCDR 2 of 9 and LCDR 3 of SEQ ID NO: 10; and (ii) a second antigen-binding domain capable of specifically binding to FolR1. The bispecific antibody of claim 1, wherein the VH of the first antigen binding domain comprises at least about 95%, 96%, 97%, 98%, 99% or 100% of the amino acid sequence of SEQ ID NO: 7 % identical amino acid sequence, and/or the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 11 . A bispecific antibody that binds to CD3 and FolR1, wherein the bispecific antibody comprises (i) a first antigen-binding domain capable of specifically binding to CD3, the first antigen-binding domain comprising the VH sequence of SEQ ID NO: 7 and the VL sequence of SEQ ID NO: 11; and (ii) a second antigen-binding domain capable of specifically binding to FolR1. The bispecific antibody of any one of claims 1 to 3, wherein the first antigen binding domain is a Fab molecule. The bispecific antibody of any one of claims 1 to 4, comprising an Fc domain consisting of a first subunit and a second subunit. The bispecific antibody of any one of claims 1 to 5, comprising a third antigen-binding domain capable of specifically binding to FolR1. The bispecific antibody of claim 6, wherein the second antigen binding domain and/or the third antigen binding domain when present are Fab molecules. The bispecific antibody of any one of claims 1 to 7, wherein the first antigen binding domain is a Fab molecule, wherein the variable domains VL and VH or the constant domains CL and of the Fab light chain and the Fab heavy chain CH1, in particular the variable domains VL and VH replace each other. The bispecific antibody of any one of claims 6 to 8, wherein the second antigen binding domain and, when present, the third antigen binding domain are conventional Fab molecules. The bispecific antibody of any one of claims 6 to 9, wherein the second antigen-binding domain and, when present, the third antigen-binding domain are Fab molecules, wherein in the constant domain CL, the amine at position 124 The amino acid is independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 123 is independently substituted with lysine (K), arginine (H), amino acid (R) or histidine (H) (according to Kabat numbering) substitution, and in this constant domain CH1 the amino acid at position 147 is independently glutamic (E) or aspartic (D) (according to the Kabat EU index number) and the amino acid at position 213 was independently substituted with glutamic acid (E) or aspartic acid (D) (according to the Kabat EU index number). The bispecific antibody of any one of claims 6 to 10, wherein the first antigen-binding domain and the second antigen-binding domain are optionally fused to each other via a peptide linker. The bispecific antibody of any one of claims 6 to 11, wherein the first antigen-binding domain and the second antigen-binding domain are each a Fab molecule, and (i) the second antigen-binding domain is in the Fab heavy chain The C-terminus is fused to the N-terminus of the Fab heavy chain of the first antigen-binding domain, or (ii) the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the second antigen-binding domain . The bispecific antibody of any one of claims 6 to 12, wherein the first antigen-binding domain, the second antigen-binding domain, and, when present, the third antigen-binding domain are each a Fab molecule, and the bispecific and wherein (i) the second antigen-binding domain is at the C-terminus of the Fab heavy chain and the N-terminus of the Fab heavy chain of the first antigen-binding domain fusion, and the first antigen-binding domain is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first subunit of the Fc domain, or (ii) the first antigen-binding domain is at the C-terminus of the Fab heavy chain and the The N-terminus of the Fab heavy chain of the second antigen-binding domain is fused, and the C-terminus of the second antigen-binding domain is fused to the N-terminus of the first subunit of the Fc domain at the C-terminus of the Fab heavy chain; and the third antigen-binding domain When present, the C-terminus of the Fab heavy chain is fused to the N-terminus of the second subunit of the Fc domain. The bispecific antibody of any one of claims 5 to 13, wherein the Fc domain is an IgG Fc domain, in particular _an IgGi Fc domain. The bispecific antibody of any one of claims 5 to 14, wherein the Fc domain is a human Fc domain. The bispecific antibody of any one of claims 5 to 15, wherein the Fc comprises a modification that promotes the association of the first subunit and the second subunit of the Fc domain. The bispecific antibody of any one of claims 5 to 16, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors and/or effector function. The bispecific antibody of any one of claims 6 to 17, wherein the second antigen binding domain and, when present, the third antigen binding domain comprise: VH comprising HCDR 1 of SEQ ID NO: 124, SEQ ID NO: 124 HCDR 2 of ID NO: 125 and HCDR 3 of SEQ ID NO: 126; and VL comprising LCDR 1 of SEQ ID NO: 8, LCDR 2 of SEQ ID NO: 9 and LCDR 3 of SEQ ID NO: 10. The bispecific antibody of claim 19 or 20, wherein the second antigen binding domain and, when present, the third antigen binding domain comprises: a VH comprising at least about 95% of the amino acid sequence of SEQ ID NO: 123 %, 96%, 97%, 98%, 99% or 100% identical amino acid sequence; and/or VL comprising at least about 95%, 96%, 97% amino acid sequence with the amino acid sequence of SEQ ID NO: 11 %, 98%, 99% or 100% identical amino acid sequences. An isolated polynucleotide encoding the bispecific antibody of any one of claims 1 to 19. A host cell comprising the isolated polynucleotide of claim 20. A method of producing a bispecific antibody that binds to CD3 and FolR1, comprising the steps of: (a) culturing a host cell as claimed in claim 21 under conditions suitable for expressing the bispecific antibody, and optionally (b) The bispecific antibody was recovered. A bispecific antibody that binds to CD3 and FolR1 produced by the method of claim 22. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 19 or 23 and a pharmaceutically acceptable carrier. The bispecific antibody according to any one of claims 1 to 19 or 23 or the pharmaceutical composition according to claim 24 is used as a medicament. The bispecific antibody according to any one of claims 1 to 19 or 23 or the pharmaceutical composition according to claim 24, for use in the treatment of cancer. Use of a bispecific antibody according to any one of claims 1 to 19 or 23 or a pharmaceutical composition according to claim 24 in the manufacture of a medicament. Use of a bispecific antibody according to any one of claims 1 to 19 or 23 or a pharmaceutical composition according to claim 24 in the manufacture of a medicament for the treatment of cancer. A method of treating a disease in a subject comprising administering to the subject an effective amount of a bispecific antibody as claimed in any one of claims 1 to 19 or 23 or a pharmaceutical composition as claimed in claim 24. The method of claim 29, wherein the disease is cancer. The present invention as set forth above.

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